WorldWideScience

Sample records for putative reductive dehalogenase

  1. Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation.

    Science.gov (United States)

    Payne, Karl Ap; Quezada, Carolina P; Fisher, Karl; Dunstan, Mark S; Collins, Fraser A; Sjuts, Hanno; Levy, Colin; Hay, Sam; Rigby, Stephen Ej; Leys, David

    2015-01-22

    Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria, with substrates including polychlorinated biphenyls or dioxins. Reductive dehalogenases form a distinct subfamily of cobalamin (B12)-dependent enzymes that are usually membrane associated and oxygen sensitive, hindering detailed studies. Here we report the characterization of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR (electron paramagnetic resonance) spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-cobalt bond chemistry catalysed by the other cobalamin-dependent subfamilies, we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-cobalt bond formation. This presents a new model in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

  2. Diverse Reductive Dehalogenases Are Associated with Clostridiales-Enriched Microcosms Dechlorinating 1,2-Dichloroethane

    KAUST Repository

    Merlino, Giuseppe; Balloi, Annalisa; Marzorati, Massimo; Mapelli, Francesca; Rizzi, Aurora; Lavazza, Davide; de Ferra, Francesca; Carpani, Giovanna; Daffonchio, Daniele

    2015-01-01

    The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales . Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community.

  3. Diverse Reductive Dehalogenases Are Associated with Clostridiales-Enriched Microcosms Dechlorinating 1,2-Dichloroethane

    KAUST Repository

    Merlino, Giuseppe

    2015-03-06

    The achievement of successful biostimulation of active microbiomes for the cleanup of a polluted site is strictly dependent on the knowledge of the key microorganisms equipped with the relevant catabolic genes responsible for the degradation process. In this work, we present the characterization of the bacterial community developed in anaerobic microcosms after biostimulation with the electron donor lactate of groundwater polluted with 1,2-dichloroethane (1,2-DCA). Through a multilevel analysis, we have assessed (i) the structural analysis of the bacterial community; (ii) the identification of putative dehalorespiring bacteria; (iii) the characterization of functional genes encoding for putative 1,2-DCA reductive dehalogenases (RDs). Following the biostimulation treatment, the structure of the bacterial community underwent a notable change of the main phylotypes, with the enrichment of representatives of the order Clostridiales . Through PCR targeting conserved regions within known RD genes, four novel variants of RDs previously associated with the reductive dechlorination of 1,2-DCA were identified in the metagenome of the Clostridiales-dominated bacterial community.

  4. Diversity of reductive dehalogenase genes from environmental samples and enrichment cultures identified with degenerate primer PCR screens.

    Directory of Open Access Journals (Sweden)

    Laura Audrey Hug

    2013-11-01

    Full Text Available Reductive dehalogenases are the critical enzymes for anaerobic organohalide respiration, a microbial metabolic process that has been harnessed for bioremediation efforts to resolve chlorinated solvent contamination in groundwater and is implicated in the global halogen cycle. Reductive dehalogenase sequence diversity is informative for the dechlorination potential of the site or enrichment culture. A suite of degenerate PCR primers targeting a comprehensive curated set of reductive dehalogenase genes was designed and applied to twelve DNA samples extracted from contaminated and pristine sites, as well as six enrichment cultures capable of reducing chlorinated compounds to non-toxic end-products. The amplified gene products from four environmental sites and two enrichment cultures were sequenced using Illumina HiSeq, and the reductive dehalogenase complement of each sample determined. The results indicate that the diversity of the reductive dehalogenase gene family is much deeper than is currently accounted for: one-third of the translated proteins have less than 70% pairwise amino acid identity to database sequences. Approximately 60% of the sequenced reductive dehalogenase genes were broadly distributed, being identified in four or more samples, and often in previously sequenced genomes as well. In contrast, 17% of the sequenced reductive dehalogenases were unique, present in only a single sample and bearing less than 90% pairwise amino acid identity to any previously identified proteins. Many of the broadly distributed reductive dehalogenases are uncharacterized in terms of their substrate specificity, making these intriguing targets for further biochemical experimentation. Finally, comparison of samples from a contaminated site and an enrichment culture derived from the same site eight years prior allowed examination of the effect of the enrichment process.

  5. Dehalogenation Activities and Distribution of Reductive Dehalogenase Homologous Genes in Marine Subsurface Sediments▿ †

    Science.gov (United States)

    Futagami, Taiki; Morono, Yuki; Terada, Takeshi; Kaksonen, Anna H.; Inagaki, Fumio

    2009-01-01

    Halogenated organic compounds serve as terminal electron acceptors for anaerobic respiration in a diverse range of microorganisms. Here, we report on the widespread distribution and diversity of reductive dehalogenase homologous (rdhA) genes in marine subsurface sediments. A total of 32 putative rdhA phylotypes were detected in sediments from the southeast Pacific off Peru, the eastern equatorial Pacific, the Juan de Fuca Ridge flank off Oregon, and the northwest Pacific off Japan, collected at a maximum depth of 358 m below the seafloor. In addition, significant dehalogenation activity involving 2,4,6-tribromophenol and trichloroethene was observed in sediment slurry from the Nankai Trough Forearc Basin. These results suggest that dehalorespiration is an important energy-yielding pathway in the subseafloor microbial ecosystem. PMID:19749069

  6. Purification and characterization of the 3-chloro-4-hydroxy-phenylacetate reductive dehalogenase of Desulfitobacterium hafniense

    DEFF Research Database (Denmark)

    Christensen, Nina; Ahring, Birgitte Kiær; Wohlfarth, Gert

    1998-01-01

    The membrane-bound 3-chloro-4-hydroxyphenylacetate (Cl-OHPA) reductive dehalogenase from the chlorophenol- educing anaerobe Desulfitobacterium hafniense was purified 11.3-fold to apparent homogeneity in the presence of the detergent CHAPS. The purified dehalogenase catalyzed the reductive...... dechlorination of Cl-OHPA to 4-hydroxyphenylacetate with reduced methyl viologen as the electron donor at a specific activity of 103.2 nkat/mg protein. SDS-PAGErevealed a single protein band with an apparent molecular mass of 46.5 kDa. The enzyme contained 0.68±0.2 mol corrinoid, 12.0±0.7 mol iron, and 13...

  7. Characterization of the corrinoid iron-sulfur protein tetrachloroethene reductive dehalogenase of Dehalobacter restrictus

    NARCIS (Netherlands)

    Maillard, J.; Schumacher, W.; Vazquez, F.; Regeard, C.; Hagen, W.R.; Holliger, C.

    2003-01-01

    The membrane-bound tetrachloroethene reductive dehalogenase (PCE-RDase) (PceA; EC 1.97.1.8), the terminal component of the respiratory chain of Dehalobacter restrictus, was purified 25-fold to apparent electrophoretic homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a

  8. Crystallization and preliminary X-ray studies of TON-1713 from Thermococcus onnurineus NA1, a putative member of the haloacid dehalogenase superfamily

    International Nuclear Information System (INIS)

    Le, Binh Van; Lee, Hyun Sook; Cho, Yona; Kang, Sung Gyun; Kim, Dong Young; Kim, Yang-Gyun; Kim, Kyeong Kyu

    2007-01-01

    A putative member of the haloacid dehalogenase superfamily from T. onnurineus has been expressed, purified and crystallized using 1.6 M magnesium sulfate as a precipitant. The crystals belonged to the triclinic space group P1 and diffracted to 1.8 Å resolution. The haloacid dehalogenase (HAD) protein superfamily is one of the largest enzyme families and shows hydrolytic activity towards diverse substrates. Structural analyses of enzymes belonging to the HAD family are required to elucidate the molecular basis underlying their broad substrate specificity and reaction mechanism. For this purpose, TON-1713, a hypothetical protein from Thermococcus onnurineus that is a member of the HAD superfamily, was expressed in Escherichia coli, purified and crystallized at 295 K using 1.6 M magnesium sulfate as a precipitant. X-ray diffraction data were collected to 1.8 Å resolution using a synchrotron-radiation source. The crystals belong to the triclinic space group P1, with unit-cell parameters a = 52.5, b = 65.8, c = 203.4 Å, α = 71.1, β = 79.9, γ = 74.3°

  9. Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-I

    International Nuclear Information System (INIS)

    Bogdanović, Xenia; Hesseler, Martin; Palm, Gottfried J.; Bornscheuer, Uwe T.; Hinrichs, Winfried

    2010-01-01

    The crystallization and preliminary X-ray diffraction studies of DppA from P. pacifica SIR-I are reported. DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P2 1 2 1 2. The DppA crystal diffracted X-rays to 1.9 Å resolution using an in-house X-ray generator

  10. PCB dechlorination hotspots and reductive dehalogenase genes in sediments from a contaminated wastewater lagoon.

    Science.gov (United States)

    Mattes, Timothy E; Ewald, Jessica M; Liang, Yi; Martinez, Andres; Awad, Andrew; Richards, Patrick; Hornbuckle, Keri C; Schnoor, Jerald L

    2017-08-12

    Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are distributed worldwide. Although industrial PCB production has stopped, legacy contamination can be traced to several different commercial mixtures (e.g., Aroclors in the USA). Despite their persistence, PCBs are subject to naturally occurring biodegradation processes, although the microbes and enzymes involved are poorly understood. The biodegradation potential of PCB-contaminated sediments in a wastewater lagoon located in Virginia (USA) was studied. Total PCB concentrations in sediments ranged from 6.34 to 12,700 mg/kg. PCB congener profiles in sediment sample were similar to Aroclor 1248; however, PCB congener profiles at several locations showed evidence of dechlorination. The sediment microbial community structure varied among samples but was dominated by Proteobacteria and Firmicutes. The relative abundance of putative dechlorinating Chloroflexi (including Dehalococcoides sp.) was 0.01-0.19% among the sediment samples, with Dehalococcoides sp. representing 0.6-14.8% of this group. Other possible PCB dechlorinators present included the Clostridia and the Geobacteraceae. A PCR survey for potential PCB reductive dehalogenase genes (RDases) yielded 11 sequences related to RDase genes in PCB-respiring Dehalococcoides mccartyi strain CG5 and PCB-dechlorinating D. mccartyi strain CBDB1. This is the first study to retrieve potential PCB RDase genes from unenriched PCB-contaminated sediments.

  11. Role of Dehalogenases in Aerobic Bacterial Degradation of Chlorinated Aromatic Compounds

    Directory of Open Access Journals (Sweden)

    Pankaj Kumar Arora

    2014-01-01

    Full Text Available This review was conducted to provide an overview of dehalogenases involved in aerobic biodegradation of chlorinated aromatic compounds. Additionally, biochemical and molecular characterization of hydrolytic, reductive, and oxygenolytic dehalogenases was reviewed. This review will increase our understanding of the process of dehalogenation of chlorinated aromatic compounds.

  12. Identification of a multi-protein reductive dehalogenase complex in Dehalococcoides mccartyi strain CBDB1 suggests a protein-dependent respiratory electron transport chain obviating quinone involvement

    DEFF Research Database (Denmark)

    Kublik, Anja; Deobald, Darja; Hartwig, Stefanie

    2016-01-01

    electrophoresis (BN-PAGE), gel filtration and ultrafiltration an active dehalogenating protein complex with a molecular mass of 250–270 kDa was identified. The active subunit of reductive dehalogenase (RdhA) colocalised with a complex iron-sulfur molybdoenzyme (CISM) subunit (CbdbA195) and an iron-sulfur cluster...... of the dehalogenating complex prior to membrane solubilisation. Taken together, the identification of the respiratory dehalogenase protein complex and the absence of indications for quinone participation in the respiration suggest a quinone-independent protein-based respiratory electron transfer chain in D. mccartyi....

  13. Organohalide Respiring Bacteria and Reductive Dehalogenases: Key Tools in Organohalide Bioremediation

    Directory of Open Access Journals (Sweden)

    Bat-Erdene eJugder

    2016-03-01

    Full Text Available Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.e. organohalide respiration. Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDase, which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses RDases, concentrating on those which have been purified (partially or wholly and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed.

  14. A review on non-stereospecific haloalkanoic acid dehalogenases ...

    African Journals Online (AJOL)

    Haloalkanoic acid dehalogenases remove halides from organic haloacids and have potential as bioremediation agents. DehE from Rhizobium sp. RC1, DehI from Pseudomonas putida PP3 and D,LDEX 113 from Pseudomonas sp. 113 are non-stereospecific dehalogenases that invert the configurations of D- and L- ...

  15. Biochemistry of Catabolic Reductive Dehalogenation.

    Science.gov (United States)

    Fincker, Maeva; Spormann, Alfred M

    2017-06-20

    A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.

  16. Identification of functional residues essential for dehalogenation by the non-stereospecific α-haloalkanoic acid dehalogenase from Rhizobium sp. RC1.

    Science.gov (United States)

    Hamid, Azzmer Azzar Abdul; Hamid, Tengku Haziyamin Tengku Abdul; Wahab, Roswanira Abdul; Huyop, Fahrul

    2015-03-01

    The non-stereospecific α-haloalkanoic acid dehalogenase DehE from Rhizobium sp. RC1 catalyzes the removal of the halide from α-haloalkanoic acid D,L-stereoisomers and, by doing so, converts them into hydroxyalkanoic acid L,D-stereoisomers, respectively. DehE has been extensively studied to determine its potential to act as a bioremediation agent, but its structure/function relationship has not been characterized. For this study, we explored the functional relevance of several putative active-site amino acids by site-specific mutagenesis. Ten active-site residues were mutated individually, and the dehalogenase activity of each of the 10 resulting mutants in soluble cell lysates against D- and L-2-chloropropionic acid was assessed. Interestingly, the mutants W34→A,F37→A, and S188→A had diminished activity, suggesting that these residues are functionally relevant. Notably, the D189→N mutant had no activity, which strongly implies that it is a catalytically important residue. Given our data, we propose a dehalogenation mechanism for DehE, which is the same as that suggested for other non-stereospecific α-haloalkanoic acid dehalogenases. To the best of our knowledge, this is the first report detailing a functional aspect for DehE, and our results could help pave the way for the bioengineering of haloalkanoic acid dehalogenases with improved catalytic properties. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Structural and mechanistic analysis of trans-3-chloroacrylic acid dehalogenase activity

    Energy Technology Data Exchange (ETDEWEB)

    Pegan, Scott D., E-mail: pegan@uic.edu [Center of Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago (United States); Serrano, Hector; Whitman, Christian P. [Division of Medicinal Chemistry, College of Pharmacy, The University of Texas, Austin (United States); Mesecar, Andrew D., E-mail: pegan@uic.edu [Center of Pharmaceutical Biotechnology and the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago (United States)

    2008-12-01

    The X-ray structure of a noncovalently modified trans-3-chloroacrylic acid dehalogenase with a substrate-homolog acetate bound in the active site has been determined to 1.7 Å resolution. Elucidation of catalytically important water is reported and multiple conformations of the catalytic residue αGlu52 are observed. Trans-3-chloroacrylic acid dehalogenase (CaaD) is a critical enzyme in the trans-1, 3-dichloropropene (DCP) degradation pathway in Pseudomonas pavonaceae 170. This enzyme allows bacteria to use trans-DCP, a common component in commercially produced fumigants, as a carbon source. CaaD specifically catalyzes the fourth step of the pathway by cofactor-independent dehalogenation of a vinyl carbon–halogen bond. Previous studies have reported an X-ray structure of CaaD under acidic conditions with a covalent modification of the catalytic βPro1 residue. Here, the 1.7 Å resolution X-ray structure of CaaD under neutral (pH 6.5) conditions is reported without the presence of the covalent adduct. In this new structure, a substrate-like acetate molecule is bound within the active site in a position analogous to the putative substrate-binding site. Additionally, a catalytically important water molecule was identified, consistent with previously proposed reaction schemes. Finally, flexibility of the catalytically relevant side chain αGlu52 is observed in the structure, supporting its role in the catalytic mechanism.

  18. High frequency of phylogenetically diverse reductive dehalogenase-homologous genes in deep subseafloor sedimentary metagenomes

    Directory of Open Access Journals (Sweden)

    Mikihiko eKawai

    2014-03-01

    Full Text Available Marine subsurface sediments on the Pacific margin harbor diverse microbial communities even at depths of several hundreds meters below the seafloor (mbsf or more. Previous PCR-based molecular analysis showed the presence of diverse reductive dehalogenase gene (rdhA homologs in marine subsurface sediment, suggesting that anaerobic respiration of organohalides is one of the possible energy-yielding pathways in the organic-rich sedimentary habitat. However, primer-independent molecular characterization of rdhA has remained to be demonstrated. Here, we studied the diversity and frequency of rdhA homologs by metagenomic analysis of five different depth horizons (0.8, 5.1, 18.6, 48.5 and 107.0 mbsf at Site C9001 off the Shimokita Peninsula of Japan. From all metagenomic pools, remarkably diverse rdhA-homologous sequences, some of which are affiliated with novel clusters, were observed with high frequency. As a comparison, we also examined frequency of dissimilatory sulfite reductase genes (dsrAB, key functional genes for microbial sulfate reduction. The dsrAB were also widely observed in the metagenomic pools whereas the frequency of dsrAB genes was generally smaller than that of rdhA-homologous genes. The phylogenetic composition of rdhA-homologous genes was similar among the five depth horizons. Our metagenomic data revealed that subseafloor rdhA homologs are more diverse than previously identified from PCR-based molecular studies. Spatial distribution of similar rdhA homologs across wide depositional ages indicates that the heterotrophic metabolic processes mediated by the genes can be ecologically important, functioning in the organic-rich subseafloor sedimentary biosphere.

  19. Expanding the Halohydrin Dehalogenase Enzyme Family: Identification of Novel Enzymes by Database Mining.

    Science.gov (United States)

    Schallmey, Marcus; Koopmeiners, Julia; Wells, Elizabeth; Wardenga, Rainer; Schallmey, Anett

    2014-12-01

    Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  20. Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    Jong, René M. de; Rozeboom, Henriëtte J.; Kalk, Kor H.; Tang, Lixia; Janssen, Dick B.; Dijkstra, Bauke W.

    2002-01-01

    Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop

  1. Crystallization and preliminary X-ray analysis of an enantioselective halohydrin dehalogenase from Agrobacterium radiobacter AD1

    NARCIS (Netherlands)

    de Jong, RM; Rozeboom, HJ; Kalk, KH; Tang, Lixia; Janssen, DB; Dijkstra, BW

    Halohydrin dehalogenases are key enzymes in the bacterial degradation of vicinal halopropanols and structurally related nematocides. Crystals of the enantioselective halohydrin dehalogenase HheC from Agrobacterium radiobacter AD1 have been obtained at room temperature from hanging-drop

  2. Kinetic mechanism and enantioselectivity of halohydrin dehalogenase from Agrobacterium radiobacter

    NARCIS (Netherlands)

    Tang, Lixia; Lutje Spelberg, Jeffrey H.; Fraaije, Marco W.; Janssen, DB

    2003-01-01

    Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the reversible intramolecular nucleophilic displacement of a halogen by a hydroxyl group in vicinal haloalcohols, producing the corresponding epoxides. The enzyme displays high enantioselectivity toward some aromatic

  3. Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications

    Directory of Open Access Journals (Sweden)

    Thiau-Fu Ang

    2018-05-01

    Full Text Available The variety of halogenated substances and their derivatives widely used as pesticides, herbicides and other industrial products is of great concern due to the hazardous nature of these compounds owing to their toxicity, and persistent environmental pollution. Therefore, from the viewpoint of environmental technology, the need for environmentally relevant enzymes involved in biodegradation of these pollutants has received a great boost. One result of this great deal of attention has been the identification of environmentally relevant bacteria that produce hydrolytic dehalogenases—key enzymes which are considered cost-effective and eco-friendly in the removal and detoxification of these pollutants. These group of enzymes catalyzing the cleavage of the carbon-halogen bond of organohalogen compounds have potential applications in the chemical industry and bioremediation. The dehalogenases make use of fundamentally different strategies with a common mechanism to cleave carbon-halogen bonds whereby, an active-site carboxylate group attacks the substrate C atom bound to the halogen atom to form an ester intermediate and a halide ion with subsequent hydrolysis of the intermediate. Structurally, these dehalogenases have been characterized and shown to use substitution mechanisms that proceed via a covalent aspartyl intermediate. More so, the widest dehalogenation spectrum of electron acceptors tested with bacterial strains which could dehalogenate recalcitrant organohalides has further proven the versatility of bacterial dehalogenators to be considered when determining the fate of halogenated organics at contaminated sites. In this review, the general features of most widely studied bacterial dehalogenases, their structural properties, basis of the degradation of organohalides and their derivatives and how they have been improved for various applications is discussed.

  4. Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications.

    Science.gov (United States)

    Ang, Thiau-Fu; Maiangwa, Jonathan; Salleh, Abu Bakar; Normi, Yahaya M; Leow, Thean Chor

    2018-05-07

    The variety of halogenated substances and their derivatives widely used as pesticides, herbicides and other industrial products is of great concern due to the hazardous nature of these compounds owing to their toxicity, and persistent environmental pollution. Therefore, from the viewpoint of environmental technology, the need for environmentally relevant enzymes involved in biodegradation of these pollutants has received a great boost. One result of this great deal of attention has been the identification of environmentally relevant bacteria that produce hydrolytic dehalogenases—key enzymes which are considered cost-effective and eco-friendly in the removal and detoxification of these pollutants. These group of enzymes catalyzing the cleavage of the carbon-halogen bond of organohalogen compounds have potential applications in the chemical industry and bioremediation. The dehalogenases make use of fundamentally different strategies with a common mechanism to cleave carbon-halogen bonds whereby, an active-site carboxylate group attacks the substrate C atom bound to the halogen atom to form an ester intermediate and a halide ion with subsequent hydrolysis of the intermediate. Structurally, these dehalogenases have been characterized and shown to use substitution mechanisms that proceed via a covalent aspartyl intermediate. More so, the widest dehalogenation spectrum of electron acceptors tested with bacterial strains which could dehalogenate recalcitrant organohalides has further proven the versatility of bacterial dehalogenators to be considered when determining the fate of halogenated organics at contaminated sites. In this review, the general features of most widely studied bacterial dehalogenases, their structural properties, basis of the degradation of organohalides and their derivatives and how they have been improved for various applications is discussed.

  5. Metagenome-derived haloalkane dehalogenases with novel catalytic properties

    Czech Academy of Sciences Publication Activity Database

    Kotík, Michael; Vaňáček, P.; Kuňka, A.; Prokop, Z.; Dambrovský, J.

    2017-01-01

    Roč. 101, č. 16 (2017), s. 6385-6397 ISSN 0175-7598 R&D Projects: GA ČR GAP504/10/0137; GA MŠk(CZ) LM2015047; GA MŠk(CZ) LM2015055 Institutional support: RVO:61388971 Keywords : Haloalkane dehalogenase * Metagenomic DNA * Heterologous production Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.420, year: 2016

  6. Identification of Multiple Dehalogenase Genes Involved in Tetrachloroethene-to-Ethene Dechlorination in a Dehalococcoides-Dominated Enrichment Culture

    Directory of Open Access Journals (Sweden)

    Mohamed Ismaeil

    2017-01-01

    Full Text Available Chloroethenes (CEs are widespread groundwater toxicants that are reductively dechlorinated to nontoxic ethene (ETH by members of Dehalococcoides. This study established a Dehalococcoides-dominated enrichment culture (designated “YN3” that dechlorinates tetrachloroethene (PCE to ETH with high dechlorination activity, that is, complete dechlorination of 800 μM PCE to ETH within 14 days in the presence of Dehalococcoides species at 5.7±1.9×107 copies of 16S rRNA gene/mL. The metagenome of YN3 harbored 18 rdhA genes (designated YN3rdhA1–18 encoding the catalytic subunit of reductive dehalogenase (RdhA, four of which were suggested to be involved in PCE-to-ETH dechlorination based on significant increases in their transcription in response to CE addition. The predicted proteins for two of these four genes, YN3RdhA8 and YN3RdhA16, showed 94% and 97% of amino acid similarity with PceA and VcrA, which are well known to dechlorinate PCE to trichloroethene (TCE and TCE to ETH, respectively. The other two rdhAs, YN3rdhA6 and YN3rdhA12, which were never proved as rdhA for CEs, showed particularly high transcription upon addition of vinyl chloride (VC, with 75±38 and 16±8.6 mRNA copies per gene, respectively, suggesting their possible functions as novel VC-reductive dehalogenases. Moreover, metagenome data indicated the presence of three coexisting bacterial species, including novel species of the genus Bacteroides, which might promote CE dechlorination by Dehalococcoides.

  7. Characterization of the haloacid dehalogenase from Xanthobacter autotrophicus GJ10 and sequencing of the dhlB gene

    DEFF Research Database (Denmark)

    van der Ploeg, J; Van Hall, Gerrit; Janssen, D B

    1991-01-01

    B) was cloned and could be allocated to a 6.5-kb EcoRI-BglII fragment. Part of this fragment was sequenced, and the dhlB open reading frame was identified by comparison with the N-terminal amino acid sequence of the protein. The gene was found to encode a protein of 27,433 Da that showed considerable homology...... chromatography. The enzyme was active with 2-halogenated carboxylic acids and converted only the L-isomer of 2-chloropropionic acid with inversion of configuration to produce D-lactate. The activity of the enzyme was not readily influenced by thiol reagents. The gene encoding the haloacid dehalogenase (dhl...... (60.5 and 61.0% similarity) with the two other haloacid dehalogenases sequenced to date but not with the haloalkane dehalogenase from X. autotrophicus GJ10....

  8. Haloalkane hydrolysis with an immobilized haloalkane dehalogenase.

    Science.gov (United States)

    Dravis, B C; Swanson, P E; Russell, A J

    2001-11-20

    Haloalkane dehalogenase from Rhodococcus rhodochrous was covalently immobilized onto a polyethyleneimine impregnated gamma-alumina support. The dehalogenating enzyme was found to retain greater than 40% of its original activity after immobilization, displaying an optimal loading (max. activity/supported protein) of 70 to 75 mg/g with an apparent maximum (max. protein/support) of 156 mg/g. The substrate, 1,2,3-trichloropropane, was found to favorably partition (adsorb) onto the inorganic alumina carrier (10 to 20 mg/g), thereby increasing the local reactant concentration with respect to the catalyst's environment, whereas the product, 2,3-dichloropropan-1-ol, demonstrated no affinity. Additionally, the inorganic alumina support exhibited no adverse effects because of solvent/component incompatibilities or deterioration due to pH variance (pH 7.0 to 10.5). As a result of the large surface area to volume ratio of the support matrix and the accessibility of the bound protein, the immobilized biocatalyst was not subject to internal mass transfer limitations. External diffusional restrictions could be eliminated with simple agitation (mixing speed: 50 rpm; flux: 4.22 cm/min). The pH-dependence of the immobilized dehalogenase was essentially the same as that for the native enzyme. Finally, both the thermostability and resistance toward inactivation by organic solvent were improved by more than an order of magnitude after immobilization. Copyright 2001 John Wiley & Sons, Inc.

  9. Concurrent Haloalkanoate Degradation and Chlorate Reduction by Pseudomonas chloritidismutans AW-1T.

    Science.gov (United States)

    Peng, Peng; Zheng, Ying; Koehorst, Jasper J; Schaap, Peter J; Stams, Alfons J M; Smidt, Hauke; Atashgahi, Siavash

    2017-06-15

    Haloalkanoates are environmental pollutants that can be degraded aerobically by microorganisms producing hydrolytic dehalogenases. However, there is a lack of information about the anaerobic degradation of haloalkanoates. Genome analysis of Pseudomonas chloritidismutans AW-1 T , a facultative anaerobic chlorate-reducing bacterium, showed the presence of two putative haloacid dehalogenase genes, the l-DEX gene and dehI , encoding an l-2-haloacid dehalogenase (l-DEX) and a halocarboxylic acid dehydrogenase (DehI), respectively. Hence, we studied the concurrent degradation of haloalkanoates and chlorate as a yet-unexplored trait of strain AW-1 T The deduced amino acid sequences of l-DEX and DehI revealed 33 to 37% and 26 to 86% identities with biochemically/structurally characterized l-DEX and the d- and dl-2-haloacid dehalogenase enzymes, respectively. Physiological experiments confirmed that strain AW-1 T can grow on chloroacetate, bromoacetate, and both l- and d-α-halogenated propionates with chlorate as an electron acceptor. Interestingly, growth and haloalkanoate degradation were generally faster with chlorate as an electron acceptor than with oxygen as an electron acceptor. In line with this, analyses of l-DEX and DehI dehalogenase activities using cell-free extract (CFE) of strain AW-1 T grown on dl-2-chloropropionate under chlorate-reducing conditions showed up to 3.5-fold higher dehalogenase activity than the CFE obtained from AW-1 T cells grown on dl-2-chloropropionate under aerobic conditions. Reverse transcription-quantitative PCR showed that the l-DEX gene was expressed constitutively independently of the electron donor (haloalkanoates or acetate) or acceptor (chlorate or oxygen), whereas the expression of dehI was induced by haloalkanoates. Concurrent degradation of organic and inorganic halogenated compounds by strain AW-1 T represents a unique metabolic capacity in a single bacterium, providing a new piece of the puzzle of the microbial halogen cycle

  10. Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene.

    Science.gov (United States)

    Bosma, Tjibbe; Damborský, Jirí; Stucki, Gerhard; Janssen, Dick B

    2002-07-01

    Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, prohibiting the development of a biological process for groundwater treatment. The critical step in the aerobic degradation of TCP is the initial dehalogenation to 2,3-dichloro-1-propanol. We used random mutagenesis and screening on eosin-methylene blue agar plates to improve the activity on TCP of the haloalkane dehalogenase from Rhodococcus sp. m15-3 (DhaA). A second-generation mutant containing two amino acid substitutions, Cys176Tyr and Tyr273Phe, was nearly eight times more efficient in dehalogenating TCP than wild-type dehalogenase. Molecular modeling of the mutant dehalogenase indicated that the Cys176Tyr mutation has a global effect on the active-site structure, allowing a more productive binding of TCP within the active site, which was further fine tuned by Tyr273Phe. The evolved haloalkane dehalogenase was expressed under control of a constitutive promoter in the 2,3-dichloro-1-propanol-utilizing bacterium Agrobacterium radiobacter AD1, and the resulting strain was able to utilize TCP as the sole carbon and energy source. These results demonstrated that directed evolution of a key catabolic enzyme and its subsequent recruitment by a suitable host organism can be used for the construction of bacteria for the degradation of a toxic and environmentally recalcitrant chemical.

  11. Compositional profile of α/β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-01-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. PMID:25171437

  12. Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry▿

    OpenAIRE

    Monincová, Marta; Prokop, Zbyněk; Vévodová, Jitka; Nagata, Yuji; Damborský, Jiří

    2007-01-01

    1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (kcat = 0.005 s−1) of Li...

  13. Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase from Pseudomonas putida strain PP3

    Energy Technology Data Exchange (ETDEWEB)

    Schmidberger, Jason W. [School of Pharmacology and Medicine, University of Western Australia, Crawley, Western Australia (Australia); Wilce, Jackie A. [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria (Australia); Weightman, Andrew J. [School of Biosciences, Cardiff University, Cardiff,Wales (United Kingdom); Wilce, Matthew C. J., E-mail: matthew.wilce@med.monash.edu.au [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria (Australia); School of Pharmacology and Medicine, University of Western Australia, Crawley, Western Australia (Australia)

    2008-07-01

    The α-haloacid dehalogenase DehI from P. putida strain PP3 was cloned into a vector with an N-terminal His tag and expressed in E. coli Nova Blue strain. Purified protein was crystallized in a primitive monoclinic form and a complete native data set was collected and analysed. Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II α-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from d-haloalkanoic acids and in some cases also the l-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I α-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2{sub 1}), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 Å, α = 90, β = 93.7, γ = 90°, and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary.

  14. Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase from Pseudomonas putida strain PP3

    International Nuclear Information System (INIS)

    Schmidberger, Jason W.; Wilce, Jackie A.; Weightman, Andrew J.; Wilce, Matthew C. J.

    2008-01-01

    The α-haloacid dehalogenase DehI from P. putida strain PP3 was cloned into a vector with an N-terminal His tag and expressed in E. coli Nova Blue strain. Purified protein was crystallized in a primitive monoclinic form and a complete native data set was collected and analysed. Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II α-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from d-haloalkanoic acids and in some cases also the l-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I α-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2 1 ), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 Å, α = 90, β = 93.7, γ = 90°, and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary

  15. Bacterial diversity and reductive dehalogenase redundancy in a 1,2-dichloroethane-degrading bacterial consortium enriched from a contaminated aquifer

    Directory of Open Access Journals (Sweden)

    Wittebolle Lieven

    2010-02-01

    Full Text Available Abstract Background Bacteria possess a reservoir of metabolic functionalities ready to be exploited for multiple purposes. The use of microorganisms to clean up xenobiotics from polluted ecosystems (e.g. soil and water represents an eco-sustainable and powerful alternative to traditional remediation processes. Recent developments in molecular-biology-based techniques have led to rapid and accurate strategies for monitoring and identification of bacteria and catabolic genes involved in the degradation of xenobiotics, key processes to follow up the activities in situ. Results We report the characterization of the response of an enriched bacterial community of a 1,2-dichloroethane (1,2-DCA contaminated aquifer to the spiking with 5 mM lactate as electron donor in microcosm studies. After 15 days of incubation, the microbial community structure was analyzed. The bacterial 16S rRNA gene clone library showed that the most represented phylogenetic group within the consortium was affiliated with the phylum Firmicutes. Among them, known degraders of chlorinated compounds were identified. A reductive dehalogenase genes clone library showed that the community held four phylogenetically-distinct catalytic enzymes, all conserving signature residues previously shown to be linked to 1,2-DCA dehalogenation. Conclusions The overall data indicate that the enriched bacterial consortium shares the metabolic functionality between different members of the microbial community and is characterized by a high functional redundancy. These are fundamental features for the maintenance of the community's functionality, especially under stress conditions and suggest the feasibility of a bioremediation treatment with a potential prompt dehalogenation and a process stability over time.

  16. Dehalogenimonas lykanthroporepellens BL-DC-9T simultaneously transcribes many rdhA genes during organohalide respiration with 1,2-DCA, 1,2-DCP, and 1,2,3-TCP as electron acceptors.

    Science.gov (United States)

    Mukherjee, Kalpataru; Bowman, Kimberly S; Rainey, Fred A; Siddaramappa, Shivakumara; Challacombe, Jean F; Moe, William M

    2014-05-01

    The genome sequence of the organohalide-respiring bacterium Dehalogenimonas lykanthroporepellensBL-DC-9(T) contains numerous loci annotated as reductive dehalogenase homologous (rdh) genes based on inferred protein sequence identity with functional dehalogenases of other bacterial species. Many of these genes are truncated, lack adjacent regulatory elements, or lack cognate genes coding for membrane-anchoring proteins typical of the functionally characterized active reductive dehalogenases of organohalide-respiring bacteria. To investigate the expression patterns of the rdh genes in D. lykanthroporepellensBL-DC-9(T), oligonucleotide primers were designed to uniquely target 25 rdh genes present in the genome as well as four putative regulatory genes. RNA extracts from cultures of strain BL-DC-9(T) actively dechlorinating three different electron acceptors, 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane were reverse-transcribed and subjected to PCR amplification using rdh-specific primers. Nineteen rdh gene transcripts, including 13 full-length rdhA genes, six truncated rdhA genes, and five rdhA genes having cognate rdhB genes were consistently detected during the dechlorination of all three of the polychlorinated alkanes tested. Transcripts from all four of the putative regulatory genes were also consistently detected. Results reported here expand the diversity of bacteria known to simultaneously transcribe multiple rdh genes and provide insights into the transcription factors associated with rdh gene expression. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  17. Kinetics of halide release of haloalkane dehalogenase : Evidence for a slow conformational change

    NARCIS (Netherlands)

    Schanstra, JP; Janssen, DB; Schanstra, Joost P.

    1996-01-01

    Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols and halides, The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water. The active site is a hydrophobic cavity buried between the main domain and the cap domain of the

  18. Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58

    International Nuclear Information System (INIS)

    Mase, Tomoko; Yabuki, Hideya; Okai, Masahiko; Ohtsuka, Jun; Imai, Fabiana Lica; Nagata, Yuji; Tanokura, Masaru

    2012-01-01

    The haloalkane dehalogenase DatA from A. tumefaciens C58 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.70 Å resolution. Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit

  19. Directed Evolution Strategies for Enantiocomplementary Haloalkane Dehalogenases : From Chemical Waste to Enantiopure Building Blocks

    NARCIS (Netherlands)

    van Leeuwen, Jan G. E.; Wijma, Hein J.; Floor, Robert J.; van der Laan, Jan-Metske; Janssen, Dick B.

    2012-01-01

    We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or (S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active

  20. REPLACEMENT OF TRYPTOPHAN RESIDUES IN HALOALKANE DEHALOGENASE REDUCES HALIDE BINDING AND CATALYTIC ACTIVITY

    NARCIS (Netherlands)

    KENNES, C; PRIES, F; KROOSHOF, GH; BOKMA, E; Kingma, Jacob; JANSSEN, DB

    1995-01-01

    Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues

  1. Crystal Structures of Intermediates in the Dehalogenation of Haloalkanoates by L-2-Haloacid Dehalogenase

    NARCIS (Netherlands)

    Ridder, Ivo S.; Rozeboom, Henriëtte J.; Kalk, Kor H.; Dijkstra, Bauke W.

    1999-01-01

    The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C2 atom. The structure of the

  2. Insights into the molecular mechanism of dehalogenation catalyzed by D-2-haloacid dehalogenase from crystal structures.

    Science.gov (United States)

    Wang, Yayue; Feng, Yanbin; Cao, Xupeng; Liu, Yinghui; Xue, Song

    2018-01-23

    D-2-haloacid dehalogenases (D-DEXs) catalyse the hydrolytic dehalogenation of D-2-haloacids, releasing halide ions and producing the corresponding 2-hydroxyacids. A structure-guided elucidation of the catalytic mechanism of this dehalogenation reaction has not been reported yet. Here, we report the catalytic mechanism of a D-DEX, HadD AJ1 from Pseudomonas putida AJ1/23, which was elucidated by X-ray crystallographic analysis and the H 2 18 O incorporation experiment. HadD AJ1 is an α-helical hydrolase that forms a homotetramer with its monomer including two structurally axisymmetric repeats. The product-bound complex structure was trapped with L-lactic acid in the active site, which is framed by the structurally related helices between two repeats. Site-directed mutagenesis confirmed the importance of the residues lining the binding pocket in stabilizing the enzyme-substrate complex. Asp205 acts as a key catalytic residue and is responsible for activating a water molecule along with Asn131. Then, the hydroxyl group of the water molecule directly attacks the C2 atom of the substrate to release the halogen ion instead of forming an enzyme-substrate ester intermediate as observed in L-2-haloacid dehalogenases. The newly revealed structural and mechanistic information on D-DEX may inspire structure-based mutagenesis to engineer highly efficient haloacid dehalogenases.

  3. Compositional profile of α / β-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

    Science.gov (United States)

    Jiménez, Diego Javier; Dini-Andreote, Francisco; Ottoni, Júlia Ronzella; de Oliveira, Valéria Maia; van Elsas, Jan Dirk; Andreote, Fernando Dini

    2015-05-01

    The occurrence of genes encoding biotechnologically relevant α/β-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/β-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes. © 2014 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  4. A bacterial haloalkane dehalogenase gene as a negative selectable marker in Arabidopsis

    DEFF Research Database (Denmark)

    Næsted, Henrik; Fennema, M.; Hao, L.

    1999-01-01

    , including Arabidopsis, tobacco, oil seed rape and rice, do not express detectable haloalkane dehalogenase activities, and that wild-type Arabidopsis grows in the presence of DCE. In contrast, DCE applied as a volatile can be used to select on plates or in soil transgenic Arabidopsis which express dhl...

  5. Directed evolution strategies for enantiocomplementary haloalkane dehalogenases: from chemical waste to enantiopure building blocks.

    Science.gov (United States)

    van Leeuwen, Jan G E; Wijma, Hein J; Floor, Robert J; van der Laan, Jan-Metske; Janssen, Dick B

    2012-01-02

    We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or (S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active epichlorohydrins-attractive intermediates for the synthesis of enantiopure fine chemicals. A dehalogenase with improved catalytic activity but very low enantioselectivity was used as the starting point. A strategy that made optimal use of the limited capacity of the screening assay, which was based on chiral gas chromatography, was developed. We used pair-wise site-saturation mutagenesis (SSM) of all 16 noncatalytic active-site residues during the initial two rounds of evolution. The resulting best R- and S-enantioselective variants were further improved in two rounds of site-restricted mutagenesis (SRM), with incorporation of carefully selected sets of amino acids at a larger number of positions, including sites that are more distant from the active site. Finally, the most promising mutations and positions were promoted to a combinatorial library by using a multi-site mutagenesis protocol with restricted codon sets. To guide the design of partly undefined (ambiguous) codon sets for these restricted libraries we employed structural information, the results of multiple sequence alignments, and knowledge from earlier rounds. After five rounds of evolution with screening of only 5500 clones, we obtained two strongly diverged haloalkane dehalogenase variants that give access to (R)-epichlorohydrin with 90 % ee and to (S)-epichlorohydrin with 97 % ee, containing 13 and 17 mutations, respectively, around their active sites. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. In Silico Phylogenetic Analysis and Molecular Modelling Study of 2-Haloalkanoic Acid Dehalogenase Enzymes from Bacterial and Fungal Origin

    Directory of Open Access Journals (Sweden)

    Raghunath Satpathy

    2016-01-01

    Full Text Available 2-Haloalkanoic acid dehalogenase enzymes have broad range of applications, starting from bioremediation to chemical synthesis of useful compounds that are widely distributed in fungi and bacteria. In the present study, a total of 81 full-length protein sequences of 2-haloalkanoic acid dehalogenase from bacteria and fungi were retrieved from NCBI database. Sequence analysis such as multiple sequence alignment (MSA, conserved motif identification, computation of amino acid composition, and phylogenetic tree construction were performed on these primary sequences. From MSA analysis, it was observed that the sequences share conserved lysine (K and aspartate (D residues in them. Also, phylogenetic tree indicated a subcluster comprised of both fungal and bacterial species. Due to nonavailability of experimental 3D structure for fungal 2-haloalkanoic acid dehalogenase in the PDB, molecular modelling study was performed for both fungal and bacterial sources of enzymes present in the subcluster. Further structural analysis revealed a common evolutionary topology shared between both fungal and bacterial enzymes. Studies on the buried amino acids showed highly conserved Leu and Ser in the core, despite variation in their amino acid percentage. Additionally, a surface exposed tryptophan was conserved in all of these selected models.

  7. Adaptation of Xanthobacter autotrophicus GJ10 to bromoacetate due to activation and mobilization of the haloacetate dehalogenase gene by insertion element IS1247

    DEFF Research Database (Denmark)

    van der Ploeg, J; Willemsen, M; Van Hall, Gerrit

    1995-01-01

    B gene. In mutant GJ10M50, a DNA fragment (designated IS1247) had copied itself from a position on the chromosome that was not linked to the dhlB region to a site immediately upstream of dhlB, resulting in a 1,672-bp insertion. IS1247 was found to encode an open reading frame corresponding to 464 amino...... acids which showed similarity to putative transposases from two other insertion elements. In most of the other MBA-resistant mutants of GJ10, IS1247 was also present in one more copy than in the wild type, which had two copies located within 20 kb. After insertion to a site proximal to dhlB, IS1247...... was able to transpose itself together with the dhlB gene to a plasmid, without the requirement of a second insertion element being present downstream of dhlB. The results show that IS1247 causes bromoacetate resistance by overexpression and mobilization of the haloacid dehalogenase gene, which mimics steps...

  8. Activation of an Asp-124→Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine

    NARCIS (Netherlands)

    Pries, Frens; Kingma, Jacob; JANSSEN, Dick B

    1995-01-01

    Haloalkane dehalogenase hydrolyses various 1-halon-alkanes to the corresponding alcohols by covalent catalysis with formation of an alkyl-enzyme intermediate. The carboxylate function of the nucleophilic aspartate (Asp-124) that displaces the halogen during formation of the intermediate was changed

  9. Repositioning the catalytic triad aspartic acid of haloalkane dehalogenase : Effects on stability, kinetics, and structure

    NARCIS (Netherlands)

    Krooshof, Geja H.; Kwant, Edwin M.; Damborský, Jiří; Koča, Jaroslav; Janssen, Dick B.

    1997-01-01

    Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member

  10. Anaerobic reductive dechlorination of tetrachloroethene: how can dual Carbon-Chlorine isotopic measurements help elucidating the underlying reaction mechanism?

    Science.gov (United States)

    Badin, Alice; Buttet, Géraldine; Maillard, Julien; Holliger, Christof; Hunkeler, Daniel

    2014-05-01

    Chlorinated ethenes (CEs) such as tetrachloroethene (PCE) are common persistent groundwater contaminants. Among clean-up strategies applied to sites affected by such pollution, bioremediation has been considered with a growing interest as it represents a cost-effective, environmental friendly approach. This technique however sometimes leads to an incomplete and slow biodegradation of CEs resulting in an accumulation of toxic metabolites. Understanding the reaction mechanisms underlying anaerobic reductive dechlorination would thus help assessing PCE biodegradation in polluted sites. Stable isotope analysis can provide insight into reaction mechanisms. For chlorinated hydrocarbons, carbon (C) and chlorine (Cl) isotope data (δ13C and δ37Cl) tend to show a linear correlation with a slope (m ≡ ɛC/ɛCl) characteristic of the reaction mechanism [1]. This study hence aims at exploring the potential of a dual C-Cl isotope approach in the determination of the reaction mechanisms involved in PCE reductive dechlorination. C and Cl isotope fractionation were investigated during anaerobic PCE dechlorination by two bacterial consortia containing members of the Sulfurospirillum genus. The specificity in these consortia resides in the fact that they each conduct PCE reductive dechlorination catalysed by one different reductive dehalogenase, i.e. PceADCE which yields trichloroethene (TCE) and cis-dichloroethene (cDCE), and PceATCE which yields TCE only. The bulk C isotope enrichment factors were -3.6±0.3 o for PceATCE and -0.7±0.1o for PceADCE. The bulk Cl isotope enrichment factors were -1.3±0.2 o for PceATCE and -0.9±0.1 o for PceADCE. When applying the dual isotope approach, two m values of 2.7±0.1 and 0.7±0.2 were obtained for the reductive dehalogenases PceATCE and PceADCE, respectively. These results suggest that PCE can be degraded according to two different mechanisms. Furthermore, despite their highly similar protein sequences, each reductive dehalogenase seems

  11. Steady-state and pre-steady-state kinetic analysis of halopropane conversion by a Rhodococcus haloalkane dehalogenase

    NARCIS (Netherlands)

    Bosma, T; Pikkemaat, MG; Kingma, Jacob; Dijk, J; Janssen, DB

    2003-01-01

    Haloalkane dehalogenase from Rhodococcus rhodochrous NCIMB 13064 (DhaA) catalyzes the hydrolysis of carbon-halogen bonds in a wide range of haloalkanes. We examined the steady-state and pre-steady-state kinetics of halopropane conversion by DhaA to illuminate mechanistic details of the

  12. Kinetic resolution of α-bromoamides: Experimental and theoretical investigation of highly enantioselective reactions catalyzed by haloalkane dehalogenases

    NARCIS (Netherlands)

    Westerbeek, Alja; Szymanski, Wiktor; Wijma, Hein J.; Marrink, Siewert; Feringa, Ben L.; Janssen, Dick B.

    2011-01-01

    Haloalkane dehalogenases from five sources were heterologously expressed in Escherichia coli, isolated, and tested for their ability to achieve kinetic resolution of racemic alpha-bromoamides, which are important intermediates used in the preparation of bioactive compounds. To explore the substrate

  13. Purifying, cloning and characterizing a novel dehalogenase from Bacillus sp. GZT to enhance the biodegradation of 2,4,6-tribromophenol in water.

    Science.gov (United States)

    Liang, Zhishu; Li, Guiying; An, Taicheng

    2017-06-01

    2,4,6-Tribromophenol (TBP), an intermediate of brominated flame retardants, can easily release to environment and recalcitrant to degradation. Previously, Bacillus sp. GZT, a pure aerobic strain capable of simultaneously debrominating and mineralizing TBP, was successfully isolated by us. To further obtain a practical application and dig up its TBP degradation mechanism, a total of 46.7-fold purification of a novel dehalogenase with a final specific activity of 18.9 U mg -1 and a molecular mass of 63.4 kDa was achieved. Under optimal conditions (35 °C and 200 rpm), up to 80% degradation efficiencies were achieved within 120 min. Adding H 2 O 2 , NADPH, Mn 2+ and Mg 2+ promoted enzyme reaction effectively; while EDTA, methyl viologen, Ni 2+ , Cu 2+ , Ca 2+ and Fe 2+ strongly inhibited reaction activities. The debromination of TBP was catalyzed by the enzyme at a Km of 78 μM and a Vmax of 0.65 min -1  mg protein -1 , which indicated that this dehalogenase could specifically eliminate TBP with a high efficiency and stability. Based on MALDI-TOF/TOF analysis, the dehalogenase shared 98% identity with peptide ABC transporter substrate-binding protein. One open reading frame (ORF) encoding this peptide was found in Strain GZT genome, subjected to clone and expressed in Escherichia coli (E. coli) to characterize the encoding gene. Result showed that this recombinant strain could also remove as similar amount of TBP as Bacillus sp. GZT under the identical condition. Based on these results, we suggest that this newly-isolated TBP dehalogenase highlights a new approach for remediating TBP pollution. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Weak activity of haloalkane dehalogenase LinB with 1,2,3-trichloropropane revealed by X-Ray crystallography and microcalorimetry.

    Science.gov (United States)

    Monincová, Marta; Prokop, Zbynek; Vévodová, Jitka; Nagata, Yuji; Damborsky, Jirí

    2007-03-01

    1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (k(cat) = 0.005 s(-1)) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities.

  15. Weak Activity of Haloalkane Dehalogenase LinB with 1,2,3-Trichloropropane Revealed by X-Ray Crystallography and Microcalorimetry▿

    Science.gov (United States)

    Monincová, Marta; Prokop, Zbyněk; Vévodová, Jitka; Nagata, Yuji; Damborský, Jiří

    2007-01-01

    1,2,3-Trichloropropane (TCP) is a highly toxic and recalcitrant compound. Haloalkane dehalogenases are bacterial enzymes that catalyze the cleavage of a carbon-halogen bond in a wide range of organic halogenated compounds. Haloalkane dehalogenase LinB from Sphingobium japonicum UT26 has, for a long time, been considered inactive with TCP, since the reaction cannot be easily detected by conventional analytical methods. Here we demonstrate detection of the weak activity (kcat = 0.005 s−1) of LinB with TCP using X-ray crystallography and microcalorimetry. This observation makes LinB a useful starting material for the development of a new biocatalyst toward TCP by protein engineering. Microcalorimetry is proposed to be a universal method for the detection of weak enzymatic activities. Detection of these activities is becoming increasingly important for engineering novel biocatalysts using the scaffolds of proteins with promiscuous activities. PMID:17259360

  16. Expression, purification and preliminary X-ray characterization of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1

    Energy Technology Data Exchange (ETDEWEB)

    Omi, Rie [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Department of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Jitsumori, Keiji; Yamauchi, Takahiro; Ichiyama, Susumu; Kurihara, Tatsuo; Esaki, Nobuyoshi [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kamiya, Nobuo; Hirotsu, Ken, E-mail: hirotsu@sci.osaka-cu.ac.jp; Miyahara, Ikuko [Department of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585 (Japan); Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2007-07-01

    A recombinant form of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1 has been expressed in E. coli, purified and crystallized. The crystal belongs to space group P6{sub 3}. Diffraction data have been collected to 1.75 Å resolution. dl-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (dl-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of dl-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P6{sub 3}, with unit-cell parameters a = b = 186.2, c = 114.4 Å. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V{sub M} value of 4.20–2.10 Å{sup 3} Da{sup −1}. A self-rotation function revealed peaks on the χ = 180° section. X-ray data have been collected to 1.75 Å resolution.

  17. Reductive dehalogenation activity of indigenous microorganism in sediments of the Hackensack River, New Jersey.

    Science.gov (United States)

    Sohn, Seo Yean; Häggblom, Max M

    2016-07-01

    Organohalogen pollutants are of concern in many river and estuarine environments, such as the New York-New Jersey Harbor estuary and its tributaries. The Hackensack River is contaminated with various metals, hydrocarbons and halogenated organics, including polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins. In order to examine the potential for microbial reductive dechlorination by indigenous microorganisms, sediment samples were collected from five different estuarine locations along the Hackensack River. Hexachlorobenzene (HCB), hexabromobenzene (HBB), and pentachloroaniline (PCA) were selected as model organohalogen pollutants to assess anaerobic dehalogenating potential. Dechlorinating activity of HCB and PCA was observed in sediment microcosms for all sampling sites. HCB was dechlorinated via pentachlorobenzene (PeCB) and trichlorobenzene (TriCB) to dichlorobenzene (DCB). PCA was dechlorinated via tetrachloroaniline (TeCA), trichloroanilines (TriCA), and dichloroanilines (DCA) to monochloroaniline (MCA). No HBB debromination was observed over 12 months of incubation. However, with HCB as a co-substrate slow HBB debromination was observed with production of tetrabromobenzene (TeBB) and tribromobenzene (TriBB). Chloroflexi specific 16S rRNA gene PCR-DGGE followed by sequence analysis detected Dehalococcoides species in sediments of the freshwater location, but not in the estuarine site. Analysis targeting 12 putative reductive dehalogenase (rdh) genes showed that these were enriched concomitant with HCB or PCA dechlorination in freshwater sediment microcosms. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5.

    Directory of Open Access Journals (Sweden)

    Anzhang Li

    Full Text Available Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k cat/K m value toward 1,3-dibromopropane (K(m = 0.82 mM, k(cat/K(m = 16.43 mM(-1 · s(-1. DadB aggregates fast in the buffers with pH ≤ 7.0, while keeps stable in monomer form when pH ≥ 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications.

  19. Biochemical characterization of a haloalkane dehalogenase DadB from Alcanivorax dieselolei B-5.

    Science.gov (United States)

    Li, Anzhang; Shao, Zongze

    2014-01-01

    Recently, we found that Alcanivorax bacteria from various marine environments were capable of degrading halogenated alkanes. Genome sequencing of A. dieselolei B-5 revealed two putative haloalkane dehalogenase (HLD) genes, which were supposed to be involved in degradation of halogenated compounds. In this report, we confirm for the first time that the Alcanivorax bacterium encodes a truly functional HLD named DadB. An activity assay with 46 halogenated substrates indicated that DadB possesses broad substrate range and has the highest overall activity among the identified HLDs. DadB prefers brominated substrates; chlorinated alkenes; and the C2-C3 substrates, including the persistent pollutants of 1,2-dichloroethane, 1,2-dichloropropane and 1,2,3-trichloropropane. As DadB displays no detectable activity toward long-chain haloalkanes such as 1-chlorohexadecane and 1-chlorooctadecane, the degradation of them in A. dieselolei B-5 might be attributed to other enzymes. Kinetic constants were determined with 6 substrates. DadB has highest affinity and largest k cat/K m value toward 1,3-dibromopropane (K(m) = 0.82 mM, k(cat)/K(m) = 16.43 mM(-1) · s(-1)). DadB aggregates fast in the buffers with pH ≤ 7.0, while keeps stable in monomer form when pH ≥ 7.5. According to homology modeling, DadB has an open active cavity with a large access tunnel, which is supposed important for larger molecules as opposed to C2-C3 substrates. Combined with the results for other HLDs, we deduce that residue I247 plays an important role in substrate selection. These results suggest that DadB and its host, A. dieselolei B-5, are of potential use for biocatalysis and bioremediation applications.

  20. Chloromuconolactone dehalogenase ClcF of actinobacteria.

    Science.gov (United States)

    Solyanikova, Inna P; Plotnikova, Elena G; Shumkova, Ekaterina S; Robota, Irina V; Prisyazhnaya, Natalya V; Golovleva, Ludmila A

    2014-01-01

    This work investigated the distribution of the clcF gene in actinobacteria isolated from different ecotopes. The gene encodes chloromuconolactone dehalogenase (CMLD) ClcF, the enzyme found to date in only one representative of Gram-positive bacteria, Rhodococcus opacus 1CP, adapted to 2-chlorophenol (2CP). Using primers specific to the clcF gene, from the DNA matrix of rhodococcal strains closely related to species Rhodococcus wratislaviensis (P1, P12, P13, P20, G10, KT112, KT723, BO1) we obtained PCR products whose nucleotide sequences were 100% identical to that of the clcF gene from strain R. opacus 1CP. CMLDs isolated from the biomass of strains Rhodococcus spp. G10 and P1 grown on 2CP did not differ by their subunit molecular mass deduced from the known amino acid sequence of the clcF gene from the ClcF of strain R. opacus 1CP. Matrix-assisted laser dissociation/ionization time-of-flight mass spectrometry showed the presence of a peak with m/z 11,194-11,196 Da both in whole cells and in protein solutions with a ClcF activity. Thus, we have first time shown the distribution of ClcF among actinobacteria isolated from geographically distant habitats.

  1. Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31.

    Science.gov (United States)

    Lahoda, Maryna; Mesters, Jeroen R; Stsiapanava, Alena; Chaloupkova, Radka; Kuty, Michal; Damborsky, Jiri; Kuta Smatanova, Ivana

    2014-02-01

    Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme-substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme-substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

  2. Biodegradation of 1,2,3-trichloropropane through directed evolution and heterologous expression of a haloalkane dehalogenase gene

    OpenAIRE

    Bosma, Tjibbe; Damborský, Jiří; Stucki, Gerhard; Janssen, Dick B.

    2002-01-01

    Using a combined strategy of random mutagenesis of haloalkane dehalogenase and genetic engineering of a chloropropanol-utilizing bacterium, we constructed an organism that is capable of growth on 1,2,3-trichloropropane (TCP). This highly toxic and recalcitrant compound is a waste product generated from the manufacture of the industrial chemical epichlorohydrin. Attempts to select and enrich bacterial cultures that can degrade TCP from environmental samples have repeatedly been unsuccessful, p...

  3. Nitrite-mediated hydrolysis of epoxides catalyzed by halohydrin dehalogenase from Agrobacterium radiobacter AD1 : A new tool for the kinetic resolution of epoxides

    NARCIS (Netherlands)

    Hasnaoui, Ghania; Lutje Spelberg, Jeffrey H.; de Vries, Erik; Tang, Lixia; Hauer, Bernhard; Janssen, Dick B.

    2005-01-01

    Halohydrin dehalogenase obtained from Agrobacterium radiobacter AD1, has been tested for the nitrite-mediated ring opening of epoxides. This reaction mainly leads to the formation of unstable hydroxynitrite ester intermediates, which can be further hydrolyzed to the corresponding diols. This

  4. Pathways and mechanisms for product release in the engineered haloalkane dehalogenases explored using classical and random acceleration molecular dynamics simulations

    Czech Academy of Sciences Publication Activity Database

    Klvana, M.; Pavlová, M.; Koudeláková, T.; Chaloupková, R.; Dvořák, P.; Prokop, Z.; Stsiapanava, A.; Kutý, Michal; Kutá-Smatanová, Ivana; Dohnálek, Jan; Kulhánek, P.; Damborský, J.

    2009-01-01

    Roč. 392, č. 5 (2009), s. 1339-1356 ISSN 0022-2836 R&D Projects: GA MŠk(CZ) LC06010 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z60870520 Keywords : haloalkane dehalogenase * product release * random acceleration molecular dynamics Subject RIV: CD - Macromolecular Chemistry Impact factor: 3.871, year: 2009

  5. Dehalogenation of Haloalkanes by Mycobacterium tuberculosis H37Rv and Other Mycobacteria

    Science.gov (United States)

    Jesenská, Andrea; Sedlác̆ek, Ivo; Damborský, Jir̆í

    2000-01-01

    Haloalkane dehalogenases convert haloalkanes to their corresponding alcohols by a hydrolytic mechanism. To date, various haloalkane dehalogenases have been isolated from bacteria colonizing environments that are contaminated with halogenated compounds. A search of current databases with the sequences of these known haloalkane dehalogenases revealed the presence of three different genes encoding putative haloalkane dehalogenases in the genome of the human parasite Mycobacterium tuberculosis H37Rv. The ability of M. tuberculosis and several other mycobacterial strains to dehalogenate haloaliphatic compounds was therefore studied. Intact cells of M. tuberculosis H37Rv were found to dehalogenate 1-chlorobutane, 1-chlorodecane, 1-bromobutane, and 1,2-dibromoethane. Nine isolates of mycobacteria from clinical material and four strains from a collection of microorganisms were found to be capable of dehalogenating 1,2-dibromoethane. Crude extracts prepared from two of these strains, Mycobacterium avium MU1 and Mycobacterium smegmatis CCM 4622, showed broad substrate specificity toward a number of halogenated substrates. Dehalogenase activity in the absence of oxygen and the identification of primary alcohols as the products of the reaction suggest a hydrolytic dehalogenation mechanism. The presence of dehalogenases in bacterial isolates from clinical material, including the species colonizing both animal tissues and free environment, indicates a possible role of parasitic microorganisms in the distribution of degradation genes in the environment. PMID:10618227

  6. Conformational Changes Allow Processing of Bulky Substrates by a Haloalkane Dehalogenase with a Small and Buried Active Site.

    Science.gov (United States)

    Kokkonen, Piia; Bednar, David; Dockalova, Veronika; Prokop, Zbynek; Damborsky, Jiri

    2018-06-01

    Haloalkane dehalogenases catalyze the hydrolysis of halogen-carbon bonds in organic halogenated compounds and as such are of great utility as biocatalysts. The crystal structures of the haloalkane dehalogenase DhlA from the bacterium from Xanthobacter autotrophicus GJ10, specifically adapted for the conversion of the small 1,2-dichloroethane (DCE) molecule, display the smallest catalytic site (110 Å3) within this enzyme family. However, during a substrate-specificity screening, we noted that DhlA can catalyze the conversion of far bulkier substrates, such as the 4-(bromomethyl)-6,7-dimethoxy-coumarin (220 Å3). This large substrate cannot bind to DhlA without conformational alterations. These conformational changes have been previously inferred from kinetic analysis, but their structural basis has not been understood. Using molecular dynamic simulations, we demonstrate here the intrinsic flexibility of part of the cap domain that allows DhlA to accommodate bulky substrates. The simulations displayed two routes for transport of substrates to the active site, one of which requires the conformational change and which is likely the route for bulky substrates. These results provide insights into the structure-dynamics-function relationships in enzymes with deeply buried active sites. Moreover, understanding the structural basis for the molecular adaptation of DhlA to DCE introduced into the biosphere during the industrial revolution provides a valuable lesson in enzyme design by nature. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Structure Prediction, Molecular Dynamics Simulation and Docking Studies of D-Specific Dehalogenase from Rhizobium sp. RC1

    Directory of Open Access Journals (Sweden)

    Ismaila Yada Sudi

    2012-11-01

    Full Text Available Currently, there is no three-dimensional structure of D-specific dehalogenase (DehD in the protein database. We modeled DehD using ab initio technique, performed molecular dynamics (MD simulation and docking of D-2-chloropropionate (D-2CP, D-2-bromopropionate (D-2BP, monochloroacetate (MCA, monobromoacetate (MBA, 2,2-dichloropropionate (2,2-DCP, d,l-2,3-dichloropropionate (d,l-2,3-DCP, and 3-chloropropionate (3-CP into the DehD active site. The sequences of DehD and D-2-haloacid dehalogenase (HadD from Pseudomonas putida AJ1 have 15% sequence similarity. The model had 80% of the amino acid residues in the most favored region when compared to the crystal structure of DehI from Pseudomonas putida PP3. Docking analysis revealed that Arg107, Arg134 and Tyr135 interacted with D-2CP, and Glu20 activated the water molecule for hydrolytic dehalogenation. Single residue substitutions at 25–30 °C showed that polar residues of DehD were stable when substituted with nonpolar residues and showed a decrease in activity within the same temperature range. The molecular dynamics simulation of DehD and its variants showed that in R134A variant, Arg107 interacted with D-2CP, while in Y135A, Gln221 and Arg231 interacted with D-2CP. It is our emphatic belief that the new model will be useful for the rational design of DehDs with enhanced potentials.

  8. Microbial dehalogenation

    NARCIS (Netherlands)

    Janssen, DB; Oppentocht, JE; Poelarends, GJ

    Novel dehalogenases have been identified recently in various bacteria that utilise halogenated substrates. X-ray studies and sequence analysis have revealed insight into the molecular mechanisms of hydrolytic dehalogenases. Furthermore, genetic and biochemical studies have indicated that reductive

  9. Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate.

    Science.gov (United States)

    Pavlova, Martina; Klvana, Martin; Prokop, Zbynek; Chaloupkova, Radka; Banas, Pavel; Otyepka, Michal; Wade, Rebecca C; Tsuda, Masataka; Nagata, Yuji; Damborsky, Jiri

    2009-10-01

    Engineering enzymes to degrade anthropogenic compounds efficiently is challenging. We obtained Rhodococcus rhodochrous haloalkane dehalogenase mutants with up to 32-fold higher activity than wild type toward the toxic, recalcitrant anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. We identified key residues in access tunnels connecting the buried active site with bulk solvent by rational design and randomized them by directed evolution. The most active mutant has large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis. These changes apparently enhance activity with TCP by decreasing accessibility of the active site for water molecules, thereby promoting activated complex formation. Kinetic analyses confirmed that the mutations improved carbon-halogen bond cleavage and shifted the rate-limiting step to the release of products. Engineering access tunnels by combining computer-assisted protein design with directed evolution may be a valuable strategy for refining catalytic properties of enzymes with buried active sites.

  10. Cobamide-mediated enzymatic reductive dehalogenation via long-range electron transfer.

    Science.gov (United States)

    Kunze, Cindy; Bommer, Martin; Hagen, Wilfred R; Uksa, Marie; Dobbek, Holger; Schubert, Torsten; Diekert, Gabriele

    2017-07-03

    The capacity of metal-containing porphyrinoids to mediate reductive dehalogenation is implemented in cobamide-containing reductive dehalogenases (RDases), which serve as terminal reductases in organohalide-respiring microbes. RDases allow for the exploitation of halogenated compounds as electron acceptors. Their reaction mechanism is under debate. Here we report on substrate-enzyme interactions in a tetrachloroethene RDase (PceA) that also converts aryl halides. The shape of PceA's highly apolar active site directs binding of bromophenols at some distance from the cobalt and with the hydroxyl substituent towards the metal. A close cobalt-substrate interaction is not observed by electron paramagnetic resonance spectroscopy. Nonetheless, a halogen substituent para to the hydroxyl group is reductively eliminated and the path of the leaving halide is traced in the structure. Based on these findings, an enzymatic mechanism relying on a long-range electron transfer is concluded, which is without parallel in vitamin B 12 -dependent biochemistry and represents an effective mode of RDase catalysis.

  11. In-silico gene co-expression network analysis in Paracoccidioides brasiliensis with reference to haloacid dehalogenase superfamily hydrolase gene

    Directory of Open Access Journals (Sweden)

    Raghunath Satpathy

    2015-01-01

    Full Text Available Context: Paracoccidioides brasiliensis, a dimorphic fungus is the causative agent of paracoccidioidomycosis, a disease globally affecting millions of people. The haloacid dehalogenase (HAD superfamily hydrolases enzyme in the fungi, in particular, is known to be responsible in the pathogenesis by adhering to the tissue. Hence, identification of novel drug targets is essential. Aims: In-silico based identification of co-expressed genes along with HAD superfamily hydrolase in P. brasiliensis during the morphogenesis from mycelium to yeast to identify possible genes as drug targets. Materials and Methods: In total, four datasets were retrieved from the NCBI-gene expression omnibus (GEO database, each containing 4340 genes, followed by gene filtration expression of the data set. Further co-expression (CE study was performed individually and then a combination these genes were visualized in the Cytoscape 2. 8.3. Statistical Analysis Used: Mean and standard deviation value of the HAD superfamily hydrolase gene was obtained from the expression data and this value was subsequently used for the CE calculation purpose by selecting specific correlation power and filtering threshold. Results: The 23 genes that were thus obtained are common with respect to the HAD superfamily hydrolase gene. A significant network was selected from the Cytoscape network visualization that contains total 7 genes out of which 5 genes, which do not have significant protein hits, obtained from gene annotation of the expressed sequence tags by BLAST X. For all the protein PSI-BLAST was performed against human genome to find the homology. Conclusions: The gene co-expression network was obtained with respect to HAD superfamily dehalogenase gene in P. Brasiliensis.

  12. Release of Halide Ions from the Buried Active Site of the Haloalkane Dehalogenase LinB Revealed by Stopped-Flow Fluorescence Analysis and Free Energy Calculations

    Czech Academy of Sciences Publication Activity Database

    Hladílková, Jana; Prokop, Z.; Chaloupková, R.; Damborský, J.; Jungwirth, Pavel

    2013-01-01

    Roč. 117, č. 46 (2013), s. 14329-14335 ISSN 1520-6106 R&D Projects: GA ČR GBP208/12/G016 Grant - others:GA ČR(CZ) GAP207/12/0775 Program:GA Institutional support: RVO:61388963 Keywords : access tunnel * buried active site * catalytic activity * enzyme mechanism * haloalkane dehalogenase * halide ions Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.377, year: 2013

  13. Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands

    International Nuclear Information System (INIS)

    Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S.; Damborsky, Jiri; Kuta Smatanova, Ivana

    2011-01-01

    Crystals of the wild-type haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 and of its catalytically inactive variant DhaA13 were grown in the presence of various ligands and diffraction data were collected to high and atomic resolution. Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon–halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2 1 2 1 2 1 as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively

  14. Sister Dehalobacter Genomes Reveal Specialization in Organohalide Respiration and Recent Strain Differentiation Likely Driven by Chlorinated Substrates

    Directory of Open Access Journals (Sweden)

    Shuiquan eTang

    2016-02-01

    Full Text Available The genomes of two closely related Dehalobacter strains (strain CF and strain DCA were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF, 1,1,1-trichloroethane (1,1,1-TCA and 1,1-dichloroethane (1,1-DCA. The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA and strain DCA (that dechlorinates 1,1-DCA each contain 17 putative reductive dehalogenase homologous (rdh genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB, and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and greater than 99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1 and corrinoid biosynthesis pathways (strains E1 and PER-K23. The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to Dehalococcoides mccartyi

  15. Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands

    Czech Academy of Sciences Publication Activity Database

    Stsiapanava, A.; Chaloupková, R.; Fořtová, A.; Brynda, Jiří; Weiss, M.S.; Damborský, J.; Kutá-Smatanová, Ivana

    2011-01-01

    Roč. 67, - (2011), s. 253-257 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LC06010 Grant - others:GA ČR(CZ) GA310/09/1407 Program:GA Institutional research plan: CEZ:AV0Z50520514; CEZ:AV0Z60870520 Keywords : haloalkane dehalogenases * DhaA * Rhodococcus rhodochrous * microseeding * atomic resolution Subject RIV: EB - Genetics ; Molecular Biology; CD - Macromolecular Chemistry (UEK-B) Impact factor: 0.506, year: 2011

  16. The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

    Science.gov (United States)

    Prokop, Pavol

    2015-08-01

    A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

  17. Regio- and Enantioselective Sequential Dehalogenation of rac-1,3-Dibromobutane by Haloalkane Dehalogenase LinB.

    Science.gov (United States)

    Gross, Johannes; Prokop, Zbyněk; Janssen, Dick; Faber, Kurt; Hall, Mélanie

    2016-08-03

    The hydrolytic dehalogenation of rac-1,3-dibromobutane catalyzed by the haloalkane dehalogenase LinB from Sphingobium japonicum UT26 proceeds in a sequential fashion: initial formation of intermediate haloalcohols followed by a second hydrolytic step to produce the final diol. Detailed investigation of the course of the reaction revealed favored nucleophilic displacement of the sec-halogen in the first hydrolytic event with pronounced R enantioselectivity. The second hydrolysis step proceeded with a regioselectivity switch at the primary position, with preference for the S enantiomer. Because of complex competition between all eight possible reactions, intermediate haloalcohols formed with moderate to good ee ((S)-4-bromobutan-2-ol: up to 87 %). Similarly, (S)-butane-1,3-diol was formed at a maximum ee of 35 % before full hydrolysis furnished the racemic diol product. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Ten Putative Contributors to the Obesity Epidemic

    Science.gov (United States)

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  19. Reductive dehalogenation of 3,5-dibromo-4-hydroxybenzoate by an aerobic strain of Delftia sp. EOB-17.

    Science.gov (United States)

    Chen, Kai; Jian, Shanshan; Huang, Linglong; Ruan, Zhepu; Li, Shunpeng; Jiang, Jiandong

    2015-12-01

    To confirm the reductive dehalogenation ability of the aerobic strain of Delftia sp. EOB-17, finding more evidences to support the hypothesis that reductive dehalogenation may occur extensively in aerobic bacteria. Delftia sp. EOB-17, isolated from terrestrial soil contaminated with halogenated aromatic compounds, completely degraded 0.2 mM DBHB in 28 h and released two equivalents of bromides under aerobic conditions in the presence of sodium succinate. LC-MS analysis revealed that DBHB was transformed to 4-hydroxybenzoate via 3-bromo-4-hydroxybenzoate by successive reductive dehalogenation. Highly conserved DBHB-degrading genes, including reductive dehalogenase gene (bhbA3) and the extra-cytoplasmic binding receptor gene (bhbB3), were also found in strain EOB-17 by genome sequencing. The optimal temperature and pH for DBHB reductive dehalogenation activity are 30 °C and 8, respectively, and 0.1 mM Cd(2+), Cu(2+), Hg(2+) and Zn(2+) strongly inhibited dehalogenation activity. The aerobic strain of Delftia sp. EOB-17 was confirmed to reductively dehalogenate DBHB under aerobic conditions, providing another evidence to support the hypothesis that reductive dehalogenation occurs extensively in aerobic bacteria.

  20. Primers That Target Functional Genes of Organohalide-Respiring Bacteria (online first)

    NARCIS (Netherlands)

    Lu, Y.; Atashgahi, S.; Hug, L.A.; Smidt, H.

    2015-01-01

    Halogenated organic hydrocarbons are problematic environmental pollutants that can be reductively dehalogenated by organohalide-respiring bacteria (OHRB) in anoxic environments. This energy-conserving process is mediated by reductive dehalogenases (RDases). To amplify the diversity of reductive

  1. Purification, crystallization and preliminary crystallographic analysis of SMU.1108c protein from Streptococcus mutans

    International Nuclear Information System (INIS)

    Feng, Ming-Jing; Fu, Tian-Min; Liu, Xiang; Li, Lan-Fen

    2010-01-01

    SMU.1108c, a putative uncharacterized protein from S. mutans, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 Å. Streptococcus mutans SMU.1108c (KEGG database) encodes a functionally uncharacterized protein consisting of 270 amino-acid residues. This protein is predicted to have a haloacid dehalogenase hydrolase-like domain and is a homologue of haloacid dehalogenase phosphatases that catalyze phosphoryl-transfer reactions. In this work, SMU.1108c was cloned into the pET28a vector and overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified to homogeneity and crystallized using the sitting-drop vapour-diffusion method. The best crystal diffracted to 2.0 Å resolution and belonged to space group C2, with unit-cell parameters a = 77.1, b = 80.2, c = 47.9 Å, β = 99.5°

  2. How Many Conformations of Enzymes Should Be Sampled for DFT/MM Calculations? A Case Study of Fluoroacetate Dehalogenase

    Directory of Open Access Journals (Sweden)

    Yanwei Li

    2016-08-01

    Full Text Available The quantum mechanics/molecular mechanics (QM/MM method (e.g., density functional theory (DFT/MM is important in elucidating enzymatic mechanisms. It is indispensable to study “multiple” conformations of enzymes to get unbiased energetic and structural results. One challenging problem, however, is to determine the minimum number of conformations for DFT/MM calculations. Here, we propose two convergence criteria, namely the Boltzmann-weighted average barrier and the disproportionate effect, to tentatively address this issue. The criteria were tested by defluorination reaction catalyzed by fluoroacetate dehalogenase. The results suggest that at least 20 conformations of enzymatic residues are required for convergence using DFT/MM calculations. We also tested the correlation of energy barriers between small QM regions and big QM regions. A roughly positive correlation was found. This kind of correlation has not been reported in the literature. The correlation inspires us to propose a protocol for more efficient sampling. This saves 50% of the computational cost in our current case.

  3. Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature.

    Science.gov (United States)

    Chaloupkova, Radka; Prokop, Zbynek; Sato, Yukari; Nagata, Yuji; Damborsky, Jiri

    2011-08-01

    The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (T(m) = 49.1 ± 0.2 °C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 °C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters Δ(R-S)ΔH and Δ(R-S)ΔS were determined. The thermodynamic analysis revealed that the resolution of β-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of α-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC3.8.1.5 haloalkane dehalogenase. © 2011 The Authors Journal compilation © 2011 FEBS.

  4. Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands.

    Science.gov (United States)

    Stsiapanava, Alena; Chaloupkova, Radka; Fortova, Andrea; Brynda, Jiri; Weiss, Manfred S; Damborsky, Jiri; Smatanova, Ivana Kuta

    2011-02-01

    Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon-halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P2(1)2(1)2(1) as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.

  5. Production of chlorothalonil hydrolytic dehalogenase from agro-industrial wastewater and its application in raw food cleaning.

    Science.gov (United States)

    He, Qin; Xu, Xi-Hui; Zhang, Fan; Tai, Yu-Kai; Luo, Yan-Fei; He, Jian; Hong, Qing; Jiang, Jian-Dong; Yan, Xin

    2017-06-01

    To reduce the fermentation cost for industrialization of chlorothalonil hydrolytic dehalogenase (Chd), agro-industrial wastewaters including molasses, corn steep liquor (CSL) and fermentation wastewater were used to substitute for expensive carbon and nitrogen sources and fresh water for lab preparation. The results showed that molasses and CSL could replace 5% carbon source and 100% organic nitrogen source respectively to maintain the same fermentation level. Re-fermentation from raffinate of ultra-filtered fermentation wastewater could achieve 61.03% of initial Chd activity and reach 96.39% activity when cultured in a mixture of raffinate and 50% of original medium constituent. Typical raw foods were chosen to evaluate the chlorothalonil removal ability of Chd. After Chd treatment for 2 h at room temperature, 97.40 and 75.55% of 30 mg kg -1 chlorothalonil on cherry tomato and strawberry respectively and 60.29% of 50 mg kg -1 chlorothalonil on Chinese cabbage were removed. Furthermore, the residual activity of the enzyme remained at 78-82% after treatment, suggesting its potential for reuse. This study proved the cost-feasibility of large-scale production of Chd from agro-industrial wastewater and demonstrated the potential of Chd in raw food cleaning. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  6. Functional Diversity of Haloacid Dehalogenase Superfamily Phosphatases from Saccharomyces cerevisiae: BIOCHEMICAL, STRUCTURAL, AND EVOLUTIONARY INSIGHTS.

    Science.gov (United States)

    Kuznetsova, Ekaterina; Nocek, Boguslaw; Brown, Greg; Makarova, Kira S; Flick, Robert; Wolf, Yuri I; Khusnutdinova, Anna; Evdokimova, Elena; Jin, Ke; Tan, Kemin; Hanson, Andrew D; Hasnain, Ghulam; Zallot, Rémi; de Crécy-Lagard, Valérie; Babu, Mohan; Savchenko, Alexei; Joachimiak, Andrzej; Edwards, Aled M; Koonin, Eugene V; Yakunin, Alexander F

    2015-07-24

    The haloacid dehalogenase (HAD)-like enzymes comprise a large superfamily of phosphohydrolases present in all organisms. The Saccharomyces cerevisiae genome encodes at least 19 soluble HADs, including 10 uncharacterized proteins. Here, we biochemically characterized 13 yeast phosphatases from the HAD superfamily, which includes both specific and promiscuous enzymes active against various phosphorylated metabolites and peptides with several HADs implicated in detoxification of phosphorylated compounds and pseudouridine. The crystal structures of four yeast HADs provided insight into their active sites, whereas the structure of the YKR070W dimer in complex with substrate revealed a composite substrate-binding site. Although the S. cerevisiae and Escherichia coli HADs share low sequence similarities, the comparison of their substrate profiles revealed seven phosphatases with common preferred substrates. The cluster of secondary substrates supporting significant activity of both S. cerevisiae and E. coli HADs includes 28 common metabolites that appear to represent the pool of potential activities for the evolution of novel HAD phosphatases. Evolution of novel substrate specificities of HAD phosphatases shows no strict correlation with sequence divergence. Thus, evolution of the HAD superfamily combines the conservation of the overall substrate pool and the substrate profiles of some enzymes with remarkable biochemical and structural flexibility of other superfamily members. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. The Complexome of Dehalococcoides mccartyi Reveals Its Organohalide Respiration-Complex Is Modular

    Directory of Open Access Journals (Sweden)

    Katja Seidel

    2018-06-01

    Full Text Available Dehalococcoides mccartyi strain CBDB1 is a slow growing strictly anaerobic microorganism dependent on halogenated compounds as terminal electron acceptor for anaerobic respiration. Indications have been described that the membrane-bound proteinaceous organohalide respiration complex of strain CBDB1 is functional without quinone-mediated electron transfer. We here study this multi-subunit protein complex in depth in regard to participating protein subunits and interactions between the subunits using blue native gel electrophoresis coupled to mass spectrometric label-free protein quantification. Applying three different solubilization modes to detach the respiration complex from the membrane we describe different solubilization snapshots of the organohalide respiration complex. The results demonstrate the existence of a two-subunit hydrogenase module loosely binding to the rest of the complex, tight binding of the subunit HupX to OmeA and OmeB, predicted to be the two subunits of a molybdopterin-binding redox subcomplex, to form a second module, and the presence of two distinct reductive dehalogenase module variants with different sizes. In our data we obtained biochemical evidence for the specificity between a reductive dehalogenase RdhA (CbdbA80 and its membrane anchor protein RdhB (CbdbB3. We also observed weak interactions between the reductive dehalogenase and the hydrogenase module suggesting a not yet recognized contact surface between these two modules. Especially an interaction between the two integral membrane subunits OmeB and RdhB seems to promote the integrity of the complex. With the different solubilization strengths we observe successive disintegration of the complex into its subunits. The observed architecture would allow the association of different reductive dehalogenase modules RdhA/RdhB with the other two protein complex modules when the strain is growing on different electron acceptors. In the search for other respiratory

  8. Reductive tetrachloroethene dehalogenation in the presence of oxygen by Sulfurospirillum multivorans: physiological studies and proteome analysis.

    Science.gov (United States)

    Gadkari, Jennifer; Goris, Tobias; Schiffmann, Christian L; Rubick, Raffael; Adrian, Lorenz; Schubert, Torsten; Diekert, Gabriele

    2018-01-01

    Reductive dehalogenation of organohalides is carried out by organohalide-respiring bacteria (OHRB) in anoxic environments. The tetrachloroethene (PCE)-respiring Epsilonproteobacterium Sulfurospirillum multivorans is one of few OHRB able to respire oxygen. Therefore, we investigated the organism's capacity to dehalogenate PCE in the presence of oxygen, which would broaden the applicability to use S. multivorans, unlike other commonly oxygen-sensitive OHRB, for bioremediation, e.g. at oxic/anoxic interphases. Additionally, this has an impact on our understanding of the global halogen cycle. Sulfurospirillum multivorans performs dehalogenation of PCE to cis-1,2-dichloroethene at oxygen concentrations below 0.19 mg/L. The redox potential of the medium electrochemically adjusted up to +400 mV had no influence on reductive dehalogenation by S. multivorans in our experiments, suggesting that higher levels of oxygen impair PCE dechlorination by inhibiting or inactivating involved enzymes. The PCE reductive dehalogenase remained active in cell extracts of S. multivorans exposed to 0.37 mg/L oxygen for more than 96 h. Analysis of the proteome revealed that superoxide reductase and cytochrome peroxidase amounts increased with 5% oxygen in the gas phase, while the response to atmospheric oxygen concentrations involved catalase and hydrogen peroxide reductase. Taken together, our results demonstrate that reductive dehalogenation by OHRB is not limited to anoxic conditions. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Mechanism of enhanced conversion of 1,2,3-trichloropropane by mutant haloalkane dehalogenase revealed by molecular modeling

    Science.gov (United States)

    Banáš, Pavel; Otyepka, Michal; Jeřábek, Petr; Petřek, Martin; Damborský, Jiří

    2006-06-01

    1,2,3-Trichloropropane (TCP) is a highly toxic, recalcitrant byproduct of epichlorohydrin manufacture. Haloalkane dehalogenase (DhaA) from Rhodococcus sp. hydrolyses the carbon-halogen bond in various halogenated compounds including TCP, but with low efficiency ( k cat/ K m = 36 s-1 M-1). A Cys176Tyr-DhaA mutant with a threefold higher catalytic efficiency for TCP dehalogenation has been previously obtained by error-prone PCR. We have used molecular simulations and quantum mechanical calculations to elucidate the molecular mechanisms involved in the improved catalysis of the mutant, and enantioselectivity of DhaA toward TCP. The Cys176Tyr mutation modifies the protein access and export routes. Substitution of the Cys residue by the bulkier Tyr narrows the upper tunnel, making the second tunnel "slot" the preferred route. TCP can adopt two major orientations in the DhaA enzyme, in one of which the halide-stabilizing residue Asn41 forms a hydrogen bond with the terminal halogen atom of the TCP molecule, while in the other it bonds with the central halogen atom. The differences in these binding patterns explain the preferential formation of the ( R)- over the ( S)-enantiomer of 2,3-dichloropropane-1-ol in the reaction catalyzed by the enzyme.

  10. Expansion of access tunnels and active-site cavities influence activity of haloalkane dehalogenases in organic cosolvents.

    Science.gov (United States)

    Stepankova, Veronika; Khabiri, Morteza; Brezovsky, Jan; Pavelka, Antonin; Sykora, Jan; Amaro, Mariana; Minofar, Babak; Prokop, Zbynek; Hof, Martin; Ettrich, Rudiger; Chaloupkova, Radka; Damborsky, Jiri

    2013-05-10

    The use of enzymes for biocatalysis can be significantly enhanced by using organic cosolvents in the reaction mixtures. Selection of the cosolvent type and concentration range for an enzymatic reaction is challenging and requires extensive empirical testing. An understanding of protein-solvent interaction could provide a theoretical framework for rationalising the selection process. Here, the behaviour of three model enzymes (haloalkane dehalogenases) was investigated in the presence of three representative organic cosolvents (acetone, formamide, and isopropanol). Steady-state kinetics assays, molecular dynamics simulations, and time-resolved fluorescence spectroscopy were used to elucidate the molecular mechanisms of enzyme-solvent interactions. Cosolvent molecules entered the enzymes' access tunnels and active sites, enlarged their volumes with no change in overall protein structure, but surprisingly did not act as competitive inhibitors. At low concentrations, the cosolvents either enhanced catalysis by lowering K(0.5) and increasing k(cat), or caused enzyme inactivation by promoting substrate inhibition and decreasing k(cat). The induced activation and inhibition of the enzymes correlated with expansion of the active-site pockets and their occupancy by cosolvent molecules. The study demonstrates that quantitative analysis of the proportions of the access tunnels and active-sites occupied by organic solvent molecules provides the valuable information for rational selection of appropriate protein-solvent pair and effective cosolvent concentration. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. CRYSTAL STRUCTURE ANALYSIS OF A PUTATIVE OXIDOREDUCTASE FROM KLEBSIELLA PNEUMONIAE

    Energy Technology Data Exchange (ETDEWEB)

    Baig, M.; Brown, A.; Eswaramoorthy, S.; Swaminathan, S.

    2009-01-01

    Klebsiella pneumoniae, a gram-negative enteric bacterium, is found in nosocomial infections which are acquired during hospital stays for about 10% of hospital patients in the United States. The crystal structure of a putative oxidoreductase from K. pneumoniae has been determined. The structural information of this K. pneumoniae protein was used to understand its function. Crystals of the putative oxidoreductase enzyme were obtained by the sitting drop vapor diffusion method using Polyethylene glycol (PEG) 3350, Bis-Tris buffer, pH 5.5 as precipitant. These crystals were used to collect X-ray data at beam line X12C of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL). The crystal structure was determined using the SHELX program and refi ned with CNS 1.1. This protein, which is involved in the catalysis of an oxidation-reduction (redox) reaction, has an alpha/beta structure. It utilizes nicotinamide adenine dinucleotide phosphate (NADP) or nicotine adenine dinucleotide (NAD) to perform its function. This structure could be used to determine the active and co-factor binding sites of the protein, information that could help pharmaceutical companies in drug design and in determining the protein’s relationship to disease treatment such as that for pneumonia and other related pathologies.

  12. Catalytic Cycle of Haloalkane Dehalogenases Toward Unnatural Substrates Explored by Computational Modeling.

    Science.gov (United States)

    Marques, Sérgio M; Dunajova, Zuzana; Prokop, Zbynek; Chaloupkova, Radka; Brezovsky, Jan; Damborsky, Jiri

    2017-08-28

    The anthropogenic toxic compound 1,2,3-trichloropropane is poorly degradable by natural enzymes. We have previously constructed the haloalkane dehalogenase DhaA31 by focused directed evolution ( Pavlova, M. et al. Nat. Chem. Biol. 2009 , 5 , 727 - 733 ), which is 32 times more active than the wild-type enzyme and is currently the most active variant known against that substrate. Recent evidence has shown that the structural basis responsible for the higher activity of DhaA31 was poorly understood. Here we have undertaken a comprehensive computational study of the main steps involved in the biocatalytic hydrolysis of 1,2,3-trichloropropane to decipher the structural basis for such enhancements. Using molecular dynamics and quantum mechanics approaches we have surveyed (i) the substrate binding, (ii) the formation of the reactive complex, (iii) the chemical step, and (iv) the release of the products. We showed that the binding of the substrate and its transport through the molecular tunnel to the active site is a relatively fast process. The cleavage of the carbon-halogen bond was previously identified as the rate-limiting step in the wild-type. Here we demonstrate that this step was enhanced in DhaA31 due to a significantly higher number of reactive configurations of the substrate and a decrease of the energy barrier to the S N 2 reaction. C176Y and V245F were identified as the key mutations responsible for most of those improvements. The release of the alcohol product was found to be the rate-limiting step in DhaA31 primarily due to the C176Y mutation. Mutational dissection of DhaA31 and kinetic analysis of the intermediate mutants confirmed the theoretical observations. Overall, our comprehensive computational approach has unveiled mechanistic details of the catalytic cycle which will enable a balanced design of more efficient enzymes. This approach is applicable to deepen the biochemical knowledge of a large number of other systems and may contribute to robust

  13. Analysis of Hydraulic Flood Control Structure at Putat Boro River

    OpenAIRE

    Ruzziyatno, Ruhban

    2015-01-01

    Putat Boro River is one of the main drainage systems of Surakarta city which drains into Bengawan Solo river. The primary problem when flood occur is the higher water level of Bengawan Solo than Boro River and then backwater occur and inundates Putat Boro River. The objective of the study is to obtain operational method of Putat Boro River floodgate to control both inflows and outflows not only during flood but also normal condition. It also aims to know the Putat Boro rivers floodgate op...

  14. Twenty putative palmitoyl-acyl transferase genes with distinct ...

    African Journals Online (AJOL)

    There are 20 genes containing DHHC domain predicted to encode putative palmitoyltransferase in Arabidopsis thaliana genome. However, little is known about their characteristics such as genetic relationship and expression profile. Here, we present an overview of the putative PAT genes in A. thaliana focusing on their ...

  15. Improved catalytic properties of halohydrin dehalogenase by modification of the halide-binding site.

    Science.gov (United States)

    Tang, Lixia; Torres Pazmiño, Daniel E; Fraaije, Marco W; de Jong, René M; Dijkstra, Bauke W; Janssen, Dick B

    2005-05-03

    Halohydrin dehalogenase (HheC) from Agrobacterium radiobacter AD1 catalyzes the dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, resulting in the formation of the corresponding epoxide, a halide ion, and a proton. Halide release is rate-limiting during the catalytic cycle of the conversion of (R)-p-nitro-2-bromo-1-phenylethanol by the enzyme. The recent elucidation of the X-ray structure of HheC showed that hydrogen bonds between the OH group of Tyr187 and between the Odelta1 atom of Asn176 and Nepsilon1 atom of Trp249 could play a role in stabilizing the conformation of the halide-binding site. The possibility that these hydrogen bonds are important for halide binding and release was studied using site-directed mutagenesis. Steady-state kinetic studies revealed that mutant Y187F, which has lost both hydrogen bonds, has a higher catalytic activity (k(cat)) with two of the three tested substrates compared to the wild-type enzyme. Mutant W249F also shows an enhanced k(cat) value with these two substrates, as well as a remarkable increase in enantiopreference for (R)-p-nitro-2-bromo-1-phenylethanol. In case of a mutation at position 176 (N176A and N176D), a 1000-fold lower catalytic efficiency (k(cat)/K(m)) was obtained, which is mainly due to an increase of the K(m) value of the enzyme. Pre-steady-state kinetic studies showed that a burst of product formation precedes the steady state, indicating that halide release is still rate-limiting for mutants Y187F and W249F. Stopped-flow fluorescence experiments revealed that the rate of halide release is 5.6-fold higher for the Y187F mutant than for the wild-type enzyme and even higher for the W249F enzyme. Taken together, these results show that the disruption of two hydrogen bonds around the halide-binding site increases the rate of halide release and can enhance the overall catalytic activity of HheC.

  16. Global Transcriptomic and Proteomic Responses of Dehalococcoides ethenogenes Strain 195 to Fixed Nitrogen Limitation

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Patrick K. H. [University of California, Berkeley; Dill, Brian [ORNL; Louie, Tiffany S. [University of California, Berkeley; Shah, Manesh B [ORNL; Verberkmoes, Nathan C [ORNL; Andersen, Gary L. [Lawrence Berkeley National Laboratory (LBNL); Zinder, Stephen H. [Cornell University; Alvarez-Cohen, Lisa [Lawrence Berkeley National Laboratory (LBNL)

    2012-01-01

    Bacteria of the genus Dehalococcoides play an important role in the reductive dechlorination of chlorinated ethenes. A systems level approach was taken in this study to examine the global transcriptomic and proteomic responses of exponentially growing D. ethenogenes strain 195 to fixed nitrogen limitation (FNL) as dechlorination activity and cell yield both decrease during FNL. As expected, the nitrogen-fixing (nif) genes were differentially up-regulated in the transcriptome and proteome of strain 195 during FNL. Aside from the nif operon, a putative methylglyoxal synthase-encoding gene (DET1576), the product of which is predicted to catalyze the formation of the toxic electrophile methylglyoxal and implicated in the uncoupling of anabolism from catabolism in bacteria, was strongly up-regulated in the transcriptome and could potentially play a role in the observed growth inhibition during FNL. Carbon catabolism genes were generally down regulated in response to FNL and a number of transporters were differentially regulated in response to nitrogen limitation, with some playing apparent roles in nitrogen acquisition while others were associated with general stress responses. A number of genes related to the functions of nucleotide synthesis, replication, transcription, translation, and post-translational modifications were also differentially expressed. One gene coding for a putative reductive dehalogenase (DET1545) and a number coding for oxidoreductases, which have implications in energy generation and redox reactions, were also differentially regulated. Interestingly, most of the genes within the multiple integrated elements were not differentially expressed. Overall, this study elucidates the molecular responses of strain 195 to FNL and identifies differentially expressed genes that are potential biomarkers to evaluate environmental cellular nitrogen status.

  17. A putative hybrid swarm within Oonopsis foliosa (Asteraceae: Astereae)

    Science.gov (United States)

    Hughes, J.F.; Brown, G.K.

    2004-01-01

    Oo??nopsis foliosa var. foliosa and var. monocephala are endemic to short-grass steppe of southeastern Colorado and until recently were considered geographically disjunct. The only known qualitative feature separating these 2 varieties is floral head type; var. foliosa has radiate heads, whereas var. monocephala heads are discoid. Sympatry between these varieties is restricted to a small area in which a range of parental types and intermediate head morphologies is observed. We used distribution mapping, morphometric analyses, chromosome cytology, and pollen stainability to characterize the sympatric zone. Morphometrics confirms that the only discrete difference between var. foliosa and var. monocephala is radiate versus discoid heads, respectively. The outer florets of putative hybrid individuals ranged from conspicuously elongated yet radially symmetric disc-floret corollas, to elongated radially asymmetric bilabiate- or deeply cleft corollas, to stunted ray florets with appendages remnant of corolla lobes. Chromosome cytology of pollen mother cells from both putative parental varieties and a series of intermediate morphological types collected at the sympatric zone reveal evidence of translocation heterozygosity. Pollen stainability shows no significant differences in viability between the parental varieties and putative hybrids. The restricted distribution of putative hybrids to a narrow zone of sympatry between the parental types and the presence of meiotic chromosome-pairing anomalies in these intermediate plants are consistent with a hybrid origin. The high stainability of putative-hybrid pollen adds to a growing body of evidence that hybrids are not universally unfit.

  18. Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study

    OpenAIRE

    Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

    2016-01-01

    Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis ...

  19. Atomic resolution studies of haloalkane dehalogenases DhaA04, DhaA14 and DhaA15 with engineered access tunnels.

    Science.gov (United States)

    Stsiapanava, A; Dohnalek, J; Gavira, J A; Kuty, M; Koudelakova, T; Damborsky, J; Kuta Smatanova, I

    2010-09-01

    The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 is a bacterial enzyme that shows catalytic activity for the hydrolytic degradation of the highly toxic industrial pollutant 1,2,3-trichloropropane (TCP). Mutagenesis focused on the access tunnels of DhaA produced protein variants with significantly improved activity towards TCP. Three mutants of DhaA named DhaA04 (C176Y), DhaA14 (I135F) and DhaA15 (C176Y + I135F) were constructed in order to study the functional relevance of the tunnels connecting the buried active site of the protein with the surrounding solvent. All three protein variants were crystallized using the sitting-drop vapour-diffusion technique. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of DhaA14 and DhaA15 had triclinic symmetry in space group P1. The crystal structures of DhaA04, DhaA14 and DhaA15 with ligands present in the active site were solved and refined using diffraction data to 1.23, 0.95 and 1.22 A, resolution, respectively. Structural comparisons of the wild type and the three mutants suggest that the tunnels play a key role in the processes of ligand exchange between the buried active site and the surrounding solvent.

  20. Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena

    Directory of Open Access Journals (Sweden)

    P.N. Agbedanu

    2013-07-01

    Full Text Available In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs as transducers of Salmonella engulfment by the model protozoan Tetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates by Tetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymena was assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule.

  1. Reductive Dehalogenation of Brominated Phenolic Compounds by Microorganisms Associated with the Marine Sponge Aplysina aerophoba

    Science.gov (United States)

    Ahn, Young-Beom; Rhee, Sung-Keun; Fennell, Donna E.; Kerkhof, Lee J.; Hentschel, Ute; Häggblom, Max M.

    2003-01-01

    Marine sponges are natural sources of brominated organic compounds, including bromoindoles, bromophenols, and bromopyrroles, that may comprise up to 12% of the sponge dry weight. Aplysina aerophoba sponges harbor large numbers of bacteria that can amount to 40% of the biomass of the animal. We postulated that there might be mechanisms for microbially mediated degradation of these halogenated chemicals within the sponges. The capability of anaerobic microorganisms associated with the marine sponge to transform haloaromatic compounds was tested under different electron-accepting conditions (i.e., denitrifying, sulfidogenic, and methanogenic). We observed dehalogenation activity of sponge-associated microorganisms with various haloaromatics. 2-Bromo-, 3-bromo-, 4-bromo-, 2,6-dibromo-, and 2,4,6-tribromophenol, and 3,5-dibromo-4-hydroxybenzoate were reductively debrominated under methanogenic and sulfidogenic conditions with no activity observed in the presence of nitrate. Monochlorinated phenols were not transformed over a period of 1 year. Debromination of 2,4,6-tribromophenol, and 2,6-dibromophenol to 2-bromophenol was more rapid than the debromination of the monobrominated phenols. Ampicillin and chloramphenicol inhibited activity, suggesting that dehalogenation was mediated by bacteria. Characterization of the debrominating methanogenic consortia by using terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis analysis indicated that different 16S ribosomal DNA (rDNA) phylotypes were enriched on the different halogenated substrates. Sponge-associated microorganisms enriched on organobromine compounds had distinct 16S rDNA TRFLP patterns and were most closely related to the δ subgroup of the proteobacteria. The presence of homologous reductive dehalogenase gene motifs in the sponge-associated microorganisms suggested that reductive dehalogenation might be coupled to dehalorespiration. PMID:12839794

  2. Novel enzymic hydrolytic dehalogenation of a chlorinated aromatic

    International Nuclear Information System (INIS)

    Scholten, J.D.; Chang, Kaihsuan; Dunaway-Mariano, D.; Babbitt, P.C.; Charest, H.; Sylvestre, M.

    1991-01-01

    Microbial enzyme systems may be used in the biodegradation of persistent environmental pollutants. The three polypeptide components of one such system, the 4-chlorobenzoate dehalogenase system, have been isolated, and the chemical steps of the 4-hydroxybenzoate-forming reaction that they catalyze have been identified. The genes contained within a 4.5-filobase Pseudomonas sp. strain CBS3 chromosomal DNA fragment that encode dehalogenase activity were selectively expressed in transformed Escherichia coli. Oligonucleotide sequencing revealed a stretch of homology between the 57-kilodalton (kD) polypeptide and several magnesium adenosine triphosphate (MgATP)-cleaving enzymes that allowed MgATP and coenzyme A (CoA) to be identified as the dehalogenase cosubstrate and cofactor, respectively. The dehalogenase activity arises from two components, a 4-chlorobenzoate:CoA ligase-dehalogenase (an αβ dimer of the 57- and 30-kD polypeptides) and a thioesterase (the 16-kD polypeptide)

  3. Genome sequence of Desulfitobacterium hafniense DCB-2, a Gram-positive anaerobe capable of dehalogenation and metal reduction

    Directory of Open Access Journals (Sweden)

    Kim Sang-Hoon

    2012-02-01

    Full Text Available Abstract Background The genome of the Gram-positive, metal-reducing, dehalorespiring Desulfitobacterium hafniense DCB-2 was sequenced in order to gain insights into its metabolic capacities, adaptive physiology, and regulatory machineries, and to compare with that of Desulfitobacterium hafniense Y51, the phylogenetically closest strain among the species with a sequenced genome. Results The genome of Desulfitobacterium hafniense DCB-2 is composed of a 5,279,134-bp circular chromosome with 5,042 predicted genes. Genome content and parallel physiological studies support the cell's ability to fix N2 and CO2, form spores and biofilms, reduce metals, and use a variety of electron acceptors in respiration, including halogenated organic compounds. The genome contained seven reductive dehalogenase genes and four nitrogenase gene homologs but lacked the Nar respiratory nitrate reductase system. The D. hafniense DCB-2 genome contained genes for 43 RNA polymerase sigma factors including 27 sigma-24 subunits, 59 two-component signal transduction systems, and about 730 transporter proteins. In addition, it contained genes for 53 molybdopterin-binding oxidoreductases, 19 flavoprotein paralogs of the fumarate reductase, and many other FAD/FMN-binding oxidoreductases, proving the cell's versatility in both adaptive and reductive capacities. Together with the ability to form spores, the presence of the CO2-fixing Wood-Ljungdahl pathway and the genes associated with oxygen tolerance add flexibility to the cell's options for survival under stress. Conclusions D. hafniense DCB-2's genome contains genes consistent with its abilities for dehalogenation, metal reduction, N2 and CO2 fixation, anaerobic respiration, oxygen tolerance, spore formation, and biofilm formation which make this organism a potential candidate for bioremediation at contaminated sites.

  4. CLONING, SEQUENCE ANALYSIS, AND CHARACTERIZATION OF PUTATIVE BETA-LACTAMASE OF STENOTROPHOMONAS MALTOPHILIA

    Directory of Open Access Journals (Sweden)

    Chong Seng Shueh

    2012-10-01

    Full Text Available The main objective of current study was to explore the function of chromosomal putative beta-lactamase gene (smlt 0115 in clinical Stenotrophomonas maltophilia. Antibiotic susceptibility test (AST screening for current antimicrobial drugs was done and Minimum Inhibitory Concentration (MIC level towards beta-lactams was determined by E-test. Putative beta-lactamase gene of S. maltophilia was amplified via PCR, with specific primers, then cloned into pET-15 expression plasmid and transformed into Escherichia coli BL21. The gene was sequenced and analyzed. The expressed protein was purified by affinity chromatography and the kinetic assay was performed. S. maltophilia ATCC 13637 was included in this experiment. Besides, a hospital strain which exhibited resistant to a series of beta-lactams including cefepime was identified via AST and MIC, hence it was named as S2 strain and was considered in this study. Sequencing result showed that putative beta-lactamase gene obtained from ATCC 13637 and S2 strains were predicted to have cephalosporinase activity by National Center for Biotechnology Information (NCBI blast program. Differences in the sequences of both ATCC 13637 and S2 strains were found via ClustalW alignment software. Kinetic assay proved a cephalosporinase characteristic produced by E. coli BL21 clone that overexpressed the putative beta-lactamase gene cloned under the control of an external promoter. Yet, expressed protein purified from S2 strain had high catalytic activity against beta-lactam antibiotics which was 14-fold higher than expressed protein purified from ATCC 13637 strain. This study represents the characterization analysis of putative beta-lactamase gene (smlt 0115 of S. maltophilia. The presence of the respective gene in the chromosome of S. maltophilia suggested that putative beta-lactamase gene (smlt 0115 of S. maltophilia plays a role in beta-lactamase resistance.

  5. Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies.

    Science.gov (United States)

    Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Toranzos, Gary A; Marota, Isolina; Giuffra, Valentina; Cano, Raul J

    2017-11-07

    Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA) gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes) may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

  6. A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

    DEFF Research Database (Denmark)

    Johannesson, Martina; Semb, Tor Henrik; Serup, Palle

    2012-01-01

    . Thus, the purpose of this study is to describe a method whereby the in vivo functionality of DE derived from ESCs can be assessed. Methods: By directed differentiation, putative DE was derived from human and mouse ESCs. This putative DE was subsequently transplanted into the endoderm of chick embryos...... to determine any occurrence of integration. Putative DE was analyzed by gene and protein expression prior to transplantation and 48 h post transplantation. Results: Putative DE, derived from mouse and human ESCs, was successfully integrated within the chick endoderm. Endoderm-specific genes were expressed...... result show that putative DE integrates with the chick endoderm and participate in the development of the chicken gut, indicating the generation of functional DE from ESCs. This functional assay can be used to assess the generation of functional DE derived from both human and mouse ESCs and provides...

  7. Toddlers' Duration of Attention toward Putative Threat

    Science.gov (United States)

    Kiel, Elizabeth J.; Buss, Kristin A.

    2011-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk of developing anxious behavior, toddlers' attention toward a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined…

  8. Lewis Base Activation of Silyl Acetals: Iridium-Catalyzed Reductive Horner-Wadsworth-Emmons Olefination.

    Science.gov (United States)

    Dakarapu, Udaya Sree; Bokka, Apparao; Asgari, Parham; Trog, Gabriela; Hua, Yuanda; Nguyen, Hiep H; Rahman, Nawal; Jeon, Junha

    2015-12-04

    A Lewis base promoted deprotonative pronucleophile addition to silyl acetals has been developed and applied to the iridium-catalyzed reductive Horner-Wadsworth-Emmons (HWE) olefination of esters and the chemoselective reduction of the resulting enoates. Lewis base activation of silyl acetals generates putative pentacoordinate silicate acetals, which fragment into aldehydes, silanes, and alkoxides in situ. Subsequent deprotonative metalation of phosphonate esters followed by HWE with aldehydes furnishes enoates. This operationally convenient, mechanistically unique protocol converts the traditionally challenging aryl, alkenyl, and alkynyl esters to homologated enoates at room temperature within a single vessel.

  9. Identification and characterization of putative conserved IAM ...

    African Journals Online (AJOL)

    Available putative AMI sequences from a wide array of monocot and dicot plants were identified and the phylogenetic tree was constructed and analyzed. We identified in this tree, a clade that contained sequences from species across the plant kingdom suggesting that AMI is conserved and may have a primary role in plant ...

  10. Microbial reductive dehalogenation of trihalomethanes by a Dehalobacter-containing co-culture.

    Science.gov (United States)

    Zhao, Siyan; Rogers, Matthew J; He, Jianzhong

    2017-07-01

    Trihalomethanes such as chloroform and bromoform, although well-known as a prominent class of disinfection by-products, are ubiquitously distributed in the environment due to widespread industrial usage in the past decades. Chloroform and bromoform are particularly concerning, of high concentrations detected and with long half-lives up to several hundred days in soils and groundwater. In this study, we report a Dehalobacter- and Desulfovibrio-containing co-culture that exhibits dehalogenation of chloroform (~0.61 mM) to dichloromethane and bromoform (~0.67 mM) to dibromomethane within 10-15 days. This co-culture was further found to dechlorinate 1,1,1-trichloroethane (1,1,1-TCA) (~0.65 mM) to 1,1-dichloroethane within 12 days. The Dehalobacter species present in this co-culture, designated Dehalobacter sp. THM1, was found to couple growth with dehalogenation of chloroform, bromoform, and 1,1,1-TCA. Strain THM1 harbors a newly identified reductive dehalogenase (RDase), ThmA, which catalyzes chloroform, bromoform, and 1,1,1-TCA dehalogenation. Additionally, based on the sequences of thmA and other identified chloroform RDase genes, ctrA, cfrA, and tmrA, a pair of chloroform RDase gene-specific primers were designed and successfully applied to investigate the chloroform dechlorinating potential of microbial communities. The comparative analysis of chloroform RDases with tetrachloroethene RDases suggests a possible approach in predicting the substrate specificity of uncharacterized RDases in the future.

  11. Gut Microbiome and Putative Resistome of Inca and Italian Nobility Mummies

    Directory of Open Access Journals (Sweden)

    Tasha M. Santiago-Rodriguez

    2017-11-01

    Full Text Available Little is still known about the microbiome resulting from the process of mummification of the human gut. In the present study, the gut microbiota, genes associated with metabolism, and putative resistome of Inca and Italian nobility mummies were characterized by using high-throughput sequencing. The Italian nobility mummies exhibited a higher bacterial diversity as compared to the Inca mummies when using 16S ribosomal (rRNA gene amplicon sequencing, but both groups showed bacterial and fungal taxa when using shotgun metagenomic sequencing that may resemble both the thanatomicrobiome and extant human gut microbiomes. Identification of sequences associated with plants, animals, and carbohydrate-active enzymes (CAZymes may provide further insights into the dietary habits of Inca and Italian nobility mummies. Putative antibiotic-resistance genes in the Inca and Italian nobility mummies support a human gut resistome prior to the antibiotic therapy era. The higher proportion of putative antibiotic-resistance genes in the Inca compared to Italian nobility mummies may support the hypotheses that a greater exposure to the environment may result in a greater acquisition of antibiotic-resistance genes. The present study adds knowledge of the microbiome resulting from the process of mummification of the human gut, insights of ancient dietary habits, and the preserved putative human gut resistome prior the antibiotic therapy era.

  12. Low Light Availability Alters Root Exudation and Reduces Putative Beneficial Microorganisms in Seagrass Roots

    Directory of Open Access Journals (Sweden)

    Belinda C. Martin

    2018-01-01

    Full Text Available Seagrass roots host a diverse microbiome that is critical for plant growth and health. Composition of microbial communities can be regulated in part by root exudates, but the specifics of these interactions in seagrass rhizospheres are still largely unknown. As light availability controls primary productivity, reduced light may impact root exudation and consequently the composition of the root microbiome. Hence, we analyzed the influence of light availability on root exudation and community structure of the root microbiome of three co-occurring seagrass species, Halophila ovalis, Halodule uninervis and Cymodocea serrulata. Plants were grown under four light treatments in mesocosms for 2 weeks; control (100% surface irradiance (SI, medium (40% SI, low (20% SI and fluctuating light (10 days 20% and 4 days 100%. 16S rDNA amplicon sequencing revealed that microbial diversity, composition and predicted function were strongly influenced by the presence of seagrass roots, such that root microbiomes were unique to each seagrass species. Reduced light availability altered seagrass root exudation, as characterized using fluorescence spectroscopy, and altered the composition of seagrass root microbiomes with a reduction in abundance of potentially beneficial microorganisms. Overall, this study highlights the potential for above-ground light reduction to invoke a cascade of changes from alterations in root exudation to a reduction in putative beneficial microorganisms and, ultimately, confirms the importance of the seagrass root environment – a critical, but often overlooked space.

  13. Putative Risk Factors in Developmental Dyslexia: A Case-Control Study of Italian Children

    Science.gov (United States)

    Mascheretti, Sara; Marino, Cecilia; Simone, Daniela; Quadrelli, Ermanno; Riva, Valentina; Cellino, Maria Rosaria; Maziade, Michel; Brombin, Chiara; Battaglia, Marco

    2015-01-01

    Although dyslexia runs in families, several putative risk factors that cannot be immediately identified as genetic predict reading disability. Published studies analyzed one or a few risk factors at a time, with relatively inconsistent results. To assess the contribution of several putative risk factors to the development of dyslexia, we conducted…

  14. Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

    International Nuclear Information System (INIS)

    Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.; Sinning, Allan; Henegar, Jeffrey; Norcross, Erin; Chinchar, V. Gregory

    2010-01-01

    Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granular bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.

  15. Pathways and mechanisms for product release in the engineered haloalkane dehalogenases explored using classical and random acceleration molecular dynamics simulations.

    Science.gov (United States)

    Klvana, Martin; Pavlova, Martina; Koudelakova, Tana; Chaloupkova, Radka; Dvorak, Pavel; Prokop, Zbynek; Stsiapanava, Alena; Kuty, Michal; Kuta-Smatanova, Ivana; Dohnalek, Jan; Kulhanek, Petr; Wade, Rebecca C; Damborsky, Jiri

    2009-10-09

    Eight mutants of the DhaA haloalkane dehalogenase carrying mutations at the residues lining two tunnels, previously observed by protein X-ray crystallography, were constructed and biochemically characterized. The mutants showed distinct catalytic efficiencies with the halogenated substrate 1,2,3-trichloropropane. Release pathways for the two dehalogenation products, 2,3-dichloropropane-1-ol and the chloride ion, and exchange pathways for water molecules, were studied using classical and random acceleration molecular dynamics simulations. Five different pathways, denoted p1, p2a, p2b, p2c, and p3, were identified. The individual pathways showed differing selectivity for the products: the chloride ion releases solely through p1, whereas the alcohol releases through all five pathways. Water molecules play a crucial role for release of both products by breakage of their hydrogen-bonding interactions with the active-site residues and shielding the charged chloride ion during its passage through a hydrophobic tunnel. Exchange of the chloride ions, the alcohol product, and the waters between the buried active site and the bulk solvent can be realized by three different mechanisms: (i) passage through a permanent tunnel, (ii) passage through a transient tunnel, and (iii) migration through a protein matrix. We demonstrate that the accessibility of the pathways and the mechanisms of ligand exchange were modified by mutations. Insertion of bulky aromatic residues in the tunnel corresponding to pathway p1 leads to reduced accessibility to the ligands and a change in mechanism of opening from permanent to transient. We propose that engineering the accessibility of tunnels and the mechanisms of ligand exchange is a powerful strategy for modification of the functional properties of enzymes with buried active sites.

  16. Putative Lineage of Novel African Usutu Virus, Central Europe

    Centers for Disease Control (CDC) Podcasts

    2015-10-15

    Sarah Gregory reads an abridged version of "Putative Lineage of Novel African Usutu Virus, Central Europe.".  Created: 10/15/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 10/15/2015.

  17. Molecular cloning and characterization of a putative OGG_N domain ...

    African Journals Online (AJOL)

    Molecular cloning and characterization of a putative OGG_N domain from the camel, Camelus dromedarius. Farid Shokry Ataya, Mohammad Saud Alanazi, Dalia Fouad, Hehsam Mahmoud Saeed, Mohammad Bazzi ...

  18. The Putative Chemosignal Androstadienone Makes Women More Generous.

    Science.gov (United States)

    Perrotta, Valentina; Graffeo, Michele; Bonini, Nicolao; Gottfried, Jay A

    2016-06-01

    Putative human chemosignals have been shown to influence mood states and emotional processing, but the connection between these effects and higher-order cognitive processing is not well established. This study utilized an economic game (Dictator Game) to test whether androstadienone (AND), an odorous compound derived from testosterone, impacts on altruistic behavior. We predicted that the female participants would act more generously in the AND condition, exhibiting a significant interaction effect between gender and AND on Dictator Game contributions. We also expected that the presence of AND should increase the positive mood of the female participants, compared to a control odor condition and also compared to the mood of the male participants. The results confirm our hypotheses: for women the subliminal perception of AND led to larger monetary donations, compared to a control odor, and also increased positive mood. These effects were absent or significantly weaker in men. Our findings highlight the capacity of human putative chemosignals to influence emotions and higher cognitive processes - in particular the processes used in the context of economic decisions - in a gender-specific way.

  19. Characteristics of functional enrichment and gene expression level of human putative transcriptional target genes.

    Science.gov (United States)

    Osato, Naoki

    2018-01-19

    Transcriptional target genes show functional enrichment of genes. However, how many and how significantly transcriptional target genes include functional enrichments are still unclear. To address these issues, I predicted human transcriptional target genes using open chromatin regions, ChIP-seq data and DNA binding sequences of transcription factors in databases, and examined functional enrichment and gene expression level of putative transcriptional target genes. Gene Ontology annotations showed four times larger numbers of functional enrichments in putative transcriptional target genes than gene expression information alone, independent of transcriptional target genes. To compare the number of functional enrichments of putative transcriptional target genes between cells or search conditions, I normalized the number of functional enrichment by calculating its ratios in the total number of transcriptional target genes. With this analysis, native putative transcriptional target genes showed the largest normalized number of functional enrichments, compared with target genes including 5-60% of randomly selected genes. The normalized number of functional enrichments was changed according to the criteria of enhancer-promoter interactions such as distance from transcriptional start sites and orientation of CTCF-binding sites. Forward-reverse orientation of CTCF-binding sites showed significantly higher normalized number of functional enrichments than the other orientations. Journal papers showed that the top five frequent functional enrichments were related to the cellular functions in the three cell types. The median expression level of transcriptional target genes changed according to the criteria of enhancer-promoter assignments (i.e. interactions) and was correlated with the changes of the normalized number of functional enrichments of transcriptional target genes. Human putative transcriptional target genes showed significant functional enrichments. Functional

  20. ald of Mycobacterium tuberculosis Encodes both the Alanine Dehydrogenase and the Putative Glycine Dehydrogenase

    Science.gov (United States)

    Giffin, Michelle M.; Modesti, Lucia; Raab, Ronald W.; Wayne, Lawrence G.

    2012-01-01

    The putative glycine dehydrogenase of Mycobacterium tuberculosis catalyzes the reductive amination of glyoxylate to glycine but not the reverse reaction. The enzyme was purified and identified as the previously characterized alanine dehydrogenase. The Ald enzyme was expressed in Escherichia coli and had both pyruvate and glyoxylate aminating activities. The gene, ald, was inactivated in M. tuberculosis, which resulted in the loss of all activities. Both enzyme activities were found associated with the cell and were not detected in the extracellular filtrate. By using an anti-Ald antibody, the protein was localized to the cell membrane, with a smaller fraction in the cytosol. None was detected in the extracellular medium. The ald knockout strain grew without alanine or glycine and was able to utilize glycine but not alanine as a nitrogen source. Transcription of ald was induced when alanine was the sole nitrogen source, and higher levels of Ald enzyme were measured. Ald is proposed to have several functions, including ammonium incorporation and alanine breakdown. PMID:22210765

  1. Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

    DEFF Research Database (Denmark)

    Nilsson, Martin; Chiang, Wen-Chi; Fazli, Mustafa

    2011-01-01

    We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P....... putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable...

  2. Exploring universal partnerships and putative marriages as tools for ...

    African Journals Online (AJOL)

    Following upon the Supreme Court of Appeal's judgment in Butters v Mncora 2012 4 SA 1 (SCA), which broadened the criteria and consequences of universal partnerships in cohabitation relationships, this article investigates the potential of universal partnerships and putative marriages to allocate rights to share in ...

  3. A new putative deltapartitivirus recovered from Dianthus amurensis.

    Science.gov (United States)

    An, Hongliu; Tan, Guanlin; Xiong, Guihong; Li, Meirong; Fang, Shouguo; Islam, Saif Ul; Zhang, Songbai; Li, Fan

    2017-09-01

    Two double stranded RNAs (dsRNA), likely representing the genome of a novel deltapartitivirus, provisionally named carnation cryptic virus 3 (CCV3), were recovered from Dianthus amurensis. The two dsRNAs were 1,573 (dsRNA1) and 1,561 (dsRNA2) bp in size, each containing a single open reading frame (ORF) encoding a 475- and 411-aa protein, respectively. The 475-aa protein contains a conserved RNA dependent RNA polymerase (RdRp) domain which shows significant homology to RdRps of established or putative partitiviruses, particularly those belonging to the genus Deltapartitivirus. However, it shares an amino acid identity of 75% with its closest relative, the RdRp of the deltapartitivirus beet cryptic virus 2 (BCV2), and is <62% identical to the RdRps of other partitiviruses. In a phylogenetic tree constructed with RdRps of selected partitiviruses, CCV3 clustered with BCV2 and formed a well-supported monophyletic clade with known or putative deltapartitiviruses.

  4. Zinc and glutamate dehydrogenase in putative glutamatergic brain structures.

    Science.gov (United States)

    Wolf, G; Schmidt, W

    1983-01-01

    A certain topographic parallelism between the distribution of histochemically (TIMM staining) identified zinc and putative glutamatergic structures in the rat brain was demonstrated. Glutamate dehydrogenase as a zinc containing protein is in consideration to be an enzyme synthesizing transmitter glutamate. In a low concentration range externally added zinc ions (10(-9) to 10(-7) M) induced an increase in the activity of glutamate dehydrogenase (GDH) originating from rat hippocampal formation, neocortex, and cerebellum up to 142.4%. With rising molarity of Zn(II) in the incubation medium, the enzyme of hippocampal formation and cerebellum showed a biphasic course of activation. Zinc ions of a concentration higher than 10(-6) M caused a strong inhibition of GDH. The effect of Zn(II) on GDH originating from spinal ganglia and liver led only to a decrease of enzyme activity. These results are discussed in connection with a functional correlation between zinc and putatively glutamatergic system.

  5. Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

    Science.gov (United States)

    Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A; Barry, Kerrie W; Spatafora, Joseph; Grigoriev, Igor V; Martin, Francis M; Pujade-Renaud, Valérie

    2018-01-01

    Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species ( Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca , and Botrosphaeria dothidea ) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

  6. Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors

    Directory of Open Access Journals (Sweden)

    David Lopez

    2018-03-01

    Full Text Available Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca, and Botrosphaeria dothidea sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector

  7. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

    Science.gov (United States)

    Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Dynamics of antibiotic resistance genes and presence of putative pathogens during ambient temperature anaerobic digestion.

    Science.gov (United States)

    Resende, J A; Diniz, C G; Silva, V L; Otenio, M H; Bonnafous, A; Arcuri, P B; Godon, J-J

    2014-12-01

    This study was focused on evaluating the persistency of antimicrobial resistance (AR) genes and putative pathogenic bacteria in an anaerobic digesters operating at mesophilic ambient temperature, in two different year seasons: summer and winter. Abundance and dynamic of AR genes encoding resistance to macrolides (ermB), aminoglycosides (aphA2) and beta-lactams (blaTEM -1 ) and persistency of potentially pathogenic bacteria in pilot-scale anaerobic digesters were investigated. AR genes were determined in the influent and effluent in both conditions. Overall, after 60 days, reduction was observed for all evaluated genes. However, during the summer, anaerobic digestion was more related to the gene reduction as compared to winter. Persistency of potentially pathogenic bacteria was also evaluated by metagenomic analyses compared to an in-house created database. Clostridium, Acinetobacter and Stenotrophomonas were the most identified. Overall, considering the mesophilic ambient temperature during anaerobic digestion (summer and winter), a decrease in pathogenic bacteria detection through metagenomic analysis and AR genes is reported. Although the mesophilic anaerobic digestion has been efficient, the results may suggest medically important bacteria and AR genes persistency during the process. This is the first report to show AR gene dynamics and persistency of potentially pathogenic bacteria through metagenomic approach in cattle manure ambient temperature anaerobic digestion. © 2014 The Society for Applied Microbiology.

  9. Putative periodontopathic bacteria and herpesviruses in pregnant women: a case-control study.

    Science.gov (United States)

    Lu, Haixia; Zhu, Ce; Li, Fei; Xu, Wei; Tao, Danying; Feng, Xiping

    2016-06-15

    Little is known about herpesvirus and putative periodontopathic bacteria in maternal chronic periodontitis. The present case-control study aimed to explore the potential relationship between putative periodontopathic bacteria and herpesviruses in maternal chronic periodontitis.Saliva samples were collected from 36 pregnant women with chronic periodontitis (cases) and 36 pregnant women with healthy periodontal status (controls). Six putative periodontopathic bacteria (Porphyromonas gingivalis [Pg], Aggregatibacer actinomycetemcomitans [Aa], Fusobacterium nucleatum [Fn], Prevotella intermedia [Pi], Tannerella forsythia [Tf], and Treponema denticola [Td]) and three herpesviruses (Epstein-Barr virus [EBV], human cytomegalovirus [HCMV], and herpes simplex virus [HSV]) were detected. Socio-demographic data and oral health related behaviors, and salivary estradiol and progesterone levels were also collected. The results showed no significant differences in socio-demographic background, oral health related behaviors, and salivary estradiol and progesterone levels between the two groups (all P > 0.05). The detection rates of included periodontopathic microorganisms were not significantly different between the two groups (all P > 0.05), but the coinfection rate of EBV and Pg was significantly higher in the case group than in the control group (P = 0.028). EBV and Pg coinfection may promote the development of chronic periodontitis among pregnant women.

  10. Computational identification of putative cytochrome P450 genes in ...

    African Journals Online (AJOL)

    In this work, a computational study of expressed sequence tags (ESTs) of soybean was performed by data mining methods and bio-informatics tools and as a result 78 putative P450 genes were identified, including 57 new ones. These genes were classified into five clans and 20 families by sequence similarities and among ...

  11. Putative golden proportions as predictors of facial esthetics in adolescents.

    NARCIS (Netherlands)

    Kiekens, R.M.A.; Kuijpers-Jagtman, A.M.; Hof, M.A. van 't; Hof, B.E. van 't; Maltha, J.C.

    2008-01-01

    INTRODUCTION: In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. METHODS: Seventy-six adult laypeople

  12. Putative Dementia Cases Fluctuate as a Function of Mini-Mental State Examination Cut-Off Points.

    Science.gov (United States)

    Rosa, Ilka M; Henriques, Ana G; Wiltfang, Jens; da Cruz E Silva, Odete A B

    2018-01-01

    As the population ages, there is a growing need to quickly and accurately identify putative dementia cases. Many cognitive tests are available; among those commonly used are the Cognitive Dementia Rating (CDR) and the Mini-Mental Status Examination (MMSE). The aim of this work was to compare the validity and reliability of these cognitive tests in a primary care based cohort (pcb-Cohort). The MMSE and the CDR were applied to 568 volunteers in the pcb-Cohort. Distinct cut-off points for the MMSE were considered, namely MMSE 27, MMSE 24, and MMSE PT (adapted for the Portuguese population). The MMSE 27 identified the greatest number of putative dementia cases, and, as determined by the ROC curve, it was the most sensitive and specific of the MMSE cut-offs considered. Putative predictive or risk factors identified included age, literacy, depression, and diabetes mellitus (DM). DM has previously been indicated as a risk factor for dementia and Alzheimer's disease. Comparatively, the MMSE 27 cut-off has the greatest sensibility (94.9%) and specificity (66.3%) when compared to MMSE PT and MMSE 24. Upon comparing MMSE and CDR scores, the latter identified a further 146 putative dementia cases, thus permitting one to propose that in an ideal situation, both tests should be employed. This increases the likelihood of identifying putative dementia cases for subsequent follow up work, thus these cognitive tests represent important tools in patient care. Further, this is a significant study for Portuguese populations, where few of these studies have been carried out.

  13. Structural Basis of Biological Nitrile Reduction*

    Science.gov (United States)

    Chikwana, Vimbai M.; Stec, Boguslaw; Lee, Bobby W. K.; de Crécy-Lagard, Valérie; Iwata-Reuyl, Dirk; Swairjo, Manal A.

    2012-01-01

    The enzyme QueF catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to 7-aminomethyl-7-deazaguanine (preQ1), the only nitrile reduction reaction known in biology. We describe here two crystal structures of Bacillus subtilis QueF, one of the wild-type enzyme in complex with the substrate preQ0, trapped as a covalent thioimide, a putative intermediate in the reaction, and the second of the C55A mutant in complex with the substrate preQ0 bound noncovalently. The QueF enzyme forms an asymmetric tunnel-fold homodecamer of two head-to-head facing pentameric subunits, harboring 10 active sites at the intersubunit interfaces. In both structures, a preQ0 molecule is bound at eight sites, and in the wild-type enzyme, it forms a thioimide covalent linkage to the catalytic residue Cys-55. Both structural and transient kinetic data show that preQ0 binding, not thioimide formation, induces a large conformational change in and closure of the active site. Based on these data, we propose a mechanism for the activation of the Cys-55 nucleophile and subsequent hydride transfer. PMID:22787148

  14. Heterologous expression and characterization of a putative glycoside hydrolase family 43 arabinofuranosidase from Clostridium thermocellum B8

    NARCIS (Netherlands)

    Camargo, de Brenda R.; Claassens, Nico J.; Quirino, Betania Ferraz; Noronha, Eliane F.; Kengen, Servé W.M.

    2018-01-01

    An extensive list of putative cellulosomal enzymes from C. thermocellum is now available in the public databanks, however, most of these remain unvalidated with regard to their activity and expression control mechanisms. This is particularly true of those enzymes putatively involved in hemicellulose

  15. Supplementary data: Variation in the PTEN-induced putative kinase ...

    Indian Academy of Sciences (India)

    Variation in the PTEN-induced putative kinase 1 gene associated with the increase risk of type 2 diabetes in northern Chinese. Yanchun Qu, Liang Sun, Ze Yang and Ruifa Han. J. Genet. 90, 125–128. Table 1. Clinical characteristics of cases and controls. Phenotype. T2DM. Controls. P value. Age (years). 49.5 ± 11.1. 50.4 ± ...

  16. Amplification of tumor inducing putative cancer stem cells (CSCs) by vitamin A/retinol from mammary tumors

    Energy Technology Data Exchange (ETDEWEB)

    Sharma, Rohit B. [Department of Microbiology and Molecular Genetics, University of Pittsburgh, PA 15261 (United States); Wang, Qingde [Department of Surgery, University of Pittsburgh, PA 15261 (United States); Khillan, Jaspal S., E-mail: khillan@pitt.edu [Department of Microbiology and Molecular Genetics, University of Pittsburgh, PA 15261 (United States)

    2013-07-12

    Highlights: •Vitamin A supports self renewal of putative CSCs from mammary tumors. •These cells exhibit impaired retinol metabolism into retinoic acid. •CSCs from mammary tumors differentiate into mammary specific cell lineages. •The cells express mammary stem cell specific CD29 and CD49f markers. •Putative CSCs form highly metastatic tumors in NOD SCID mouse. -- Abstract: Solid tumors contain a rare population of cancer stem cells (CSCs) that are responsible for relapse and metastasis. The existence of CSC however, remains highly controversial issue. Here we present the evidence for putative CSCs from mammary tumors amplified by vitamin A/retinol signaling. The cells exhibit mammary stem cell specific CD29{sup hi}/CD49f{sup hi}/CD24{sup hi} markers, resistance to radiation and chemo therapeutic agents and form highly metastatic tumors in NOD/SCID mice. The cells exhibit indefinite self renewal as cell lines. Furthermore, the cells exhibit impaired retinol metabolism and do not express enzymes that metabolize retinol into retinoic acid. Vitamin A/retinol also amplified putative CSCs from breast cancer cell lines that form highly aggressive tumors in NOD SCID mice. The studies suggest that high purity putative CSCs can be isolated from solid tumors to establish patient specific cell lines for personalized therapeutics for pre-clinical translational applications. Characterization of CSCs will allow understanding of basic cellular and molecular pathways that are deregulated, mechanisms of tumor metastasis and evasion of therapies that has direct clinical relevance.

  17. Alkenenitrile Transmissive Olefination: Synthesis of the Putative Lignan "Morinol I"

    Science.gov (United States)

    Fleming, Fraser F.; Liu, Wang; Yao, Lihua; Pitta, Bhaskar; Purzycki, Matthew; Ravikumar, P. C.

    2012-01-01

    Grignard reagents trigger an addition-elimination with α'-hydroxy acrylonitriles to selectively generate Z-alkenenitriles. The modular assembly of Z-alkenenitriles from a Grignard reagent, acrylonitrile, and an aldehyde is ideal for stereoselectively synthesizing alkenes as illustrated in the synthesis of the putative lignan "morinol I." PMID:22545004

  18. Identification of a putative nuclear export signal motif in human NANOG homeobox domain

    International Nuclear Information System (INIS)

    Park, Sung-Won; Do, Hyun-Jin; Huh, Sun-Hyung; Sung, Boreum; Uhm, Sang-Jun; Song, Hyuk; Kim, Nam-Hyung; Kim, Jae-Hwan

    2012-01-01

    Highlights: ► We found the putative nuclear export signal motif within human NANOG homeodomain. ► Leucine-rich residues are important for human NANOG homeodomain nuclear export. ► CRM1-specific inhibitor LMB blocked the potent human NANOG NES-mediated nuclear export. -- Abstract: NANOG is a homeobox-containing transcription factor that plays an important role in pluripotent stem cells and tumorigenic cells. To understand how nuclear localization of human NANOG is regulated, the NANOG sequence was examined and a leucine-rich nuclear export signal (NES) motif ( 125 MQELSNILNL 134 ) was found in the homeodomain (HD). To functionally validate the putative NES motif, deletion and site-directed mutants were fused to an EGFP expression vector and transfected into COS-7 cells, and the localization of the proteins was examined. While hNANOG HD exclusively localized to the nucleus, a mutant with both NLSs deleted and only the putative NES motif contained (hNANOG HD-ΔNLSs) was predominantly cytoplasmic, as observed by nucleo/cytoplasmic fractionation and Western blot analysis as well as confocal microscopy. Furthermore, site-directed mutagenesis of the putative NES motif in a partial hNANOG HD only containing either one of the two NLS motifs led to localization in the nucleus, suggesting that the NES motif may play a functional role in nuclear export. Furthermore, CRM1-specific nuclear export inhibitor LMB blocked the hNANOG potent NES-mediated export, suggesting that the leucine-rich motif may function in CRM1-mediated nuclear export of hNANOG. Collectively, a NES motif is present in the hNANOG HD and may be functionally involved in CRM1-mediated nuclear export pathway.

  19. Site-Specific, Covalent Immobilization of Dehalogenase ST2570 Catalyzed by Formylglycine-Generating Enzymes and Its Application in Batch and Semi-Continuous Flow Reactors

    Directory of Open Access Journals (Sweden)

    Hui Jian

    2016-07-01

    Full Text Available Formylglycine-generating enzymes can selectively recognize and oxidize cysteine residues within the sulfatase sub motif at the terminus of proteins to form aldehyde-bearing formylglycine (FGly residues, and are normally used in protein labeling. In this study, an aldehyde tag was introduced to proteins using formylglycine-generating enzymes encoded by a reconstructed set of the pET28a plasmid system for enzyme immobilization. The haloacid dehalogenase ST2570 from Sulfolobus tokodaii was used as a model enzyme. The C-terminal aldehyde-tagged ST2570 (ST2570CQ exhibited significant enzymological properties, such as new free aldehyde groups, a high level of protein expression and improved enzyme activity. SBA-15 has widely been used as an immobilization support for its large surface and excellent thermal and chemical stability. It was functionalized with amino groups by aminopropyltriethoxysilane. The C-terminal aldehyde-tagged ST2570 was immobilized to SBA-15 by covalent binding. The site-specific immobilization of ST2570 avoided the chemical denaturation that occurs in general covalent immobilization and resulted in better fastening compared to physical adsorption. The site-specific immobilized ST2570 showed 3-fold higher thermal stability, 1.2-fold higher catalytic ability and improved operational stability than free ST2570. The site-specific immobilized ST2570 retained 60% of its original activity after seven cycles of batch operation, and it was superior to the ST2570 immobilized to SBA-15 by physical adsorption, which loses 40% of its original activity when used for the second time. It is remarkable that the site-specific immobilized ST2570 still retained 100% of its original activity after 10 cycles of reuse in the semi-continuous flow reactor. Overall, these results provide support for the industrial-scale production and application of site-specific, covalently immobilized ST2570.

  20. Search strings for the study of putative occupational determinants of disease

    NARCIS (Netherlands)

    Mattioli, Stefano; Zanardi, Francesca; Baldasseroni, Alberto; Schaafsma, Frederieke; Cooke, Robin M. T.; Mancini, Gianpiero; Fierro, Mauro; Santangelo, Chiara; Farioli, Andrea; Fucksia, Serenella; Curti, Stefania; Violante, Francesco S.; Verbeek, Jos

    2010-01-01

    To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms describing

  1. Putative Biomarkers and Targets of Estrogen Receptor Negative Human Breast Cancer

    Directory of Open Access Journals (Sweden)

    Stephen W. Byers

    2011-07-01

    Full Text Available Breast cancer is a progressive and potentially fatal disease that affects women of all ages. Like all progressive diseases, early and reliable diagnosis is the key for successful treatment and annihilation. Biomarkers serve as indicators of pathological, physiological, or pharmacological processes. Her2/neu, CA15.3, estrogen receptor (ER, progesterone receptor (PR, and cytokeratins are biomarkers that have been approved by the Food and Drug Administration for disease diagnosis, prognosis, and therapy selection. The structural and functional complexity of protein biomarkers and the heterogeneity of the breast cancer pathology present challenges to the scientific community. Here we review estrogen receptor-related putative breast cancer biomarkers, including those of putative breast cancer stem cells, a minor population of estrogen receptor negative tumor cells that retain the stem cell property of self renewal. We also review a few promising cytoskeleton targets for ER alpha negative breast cancer.

  2. Sequence analysis of putative swrW gene required for surfactant ...

    African Journals Online (AJOL)

    owner

    2012-07-17

    Jul 17, 2012 ... These nucleotide and protein sequence analysis of the putative swrW gene provides vital information on the versatility .... chain reaction (PCR) products were stored at 4°C. Presence of ... identical to the same gene with an E-value of 0.0. .... The Prokaryotes-A Handbook on the Biol. of Bacteria:Ecophysiol.

  3. A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins.

    Directory of Open Access Journals (Sweden)

    Siddarth Soni

    Full Text Available Synchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID. The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue.General membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2, Nexilin (NEXN, Popeye-domain-containg-protein 2 (POPDC2 and thioredoxin-related-transmembrane-protein 2 (TMX2 and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes.The presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart.

  4. Putative and unique gene sequence utilization for the design of species specific probes as modeled by Lactobacillus plantarum

    Science.gov (United States)

    The concept of utilizing putative and unique gene sequences for the design of species specific probes was tested. The abundance profile of assigned functions within the Lactobacillus plantarum genome was used for the identification of the putative and unique gene sequence, csh. The targeted gene (cs...

  5. Search strings for the study of putative occupational determinants of disease

    NARCIS (Netherlands)

    Mattioli, S.; Zanardi, F.; Baldasseroni, A.; Schaafsma, F.; Cooke, R.M.T.; Mancini, G.; Fierro, M.; Santangelo, C.; Farioli, A.; Fucksia, S.; Curti, S.; Violante, F.S.; Verbeek, J.

    2010-01-01

    Objective To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Methods Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms

  6. Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes

    Directory of Open Access Journals (Sweden)

    Holly L. Sewell

    2017-12-01

    Full Text Available The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi. 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4 in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2 as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi. We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA in the phylum Chloroflexi (DscP3 and Dsc4, including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187 of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of

  7. Preliminary characterization and biological reduction of putative biogenic iron oxides (BIOS) from the Tonga-Kermadec Arc, southwest Pacific Ocean.

    Science.gov (United States)

    Langley, S; Igric, P; Takahashi, Y; Sakai, Y; Fortin, D; Hannington, M D; Schwarz-Schampera, U

    2009-01-01

    Sediment samples were obtained from areas of diffuse hydrothermal venting along the seabed in the Tonga sector of the Tonga-Kermadec Arc, southwest Pacific Ocean. Sediments from Volcano 1 and Volcano 19 were analyzed by X-ray diffraction (XRD) and found to be composed primarily of the iron oxyhydroxide mineral, two-line ferrihydrite. XRD also suggested the possible presence of minor amounts of more ordered iron (hydr)oxides (including six-line ferrihydrite, goethite/lepidocrocite and magnetite) in the biogenic iron oxides (BIOS) from Volcano 1; however, Mössbauer spectroscopy failed to detect any mineral phases more crystalline than two-line ferrihydrite. The minerals were precipitated on the surfaces of abundant filamentous microbial structures. Morphologically, some of these structures were similar in appearance to the known iron-oxidizing genus Mariprofundus spp., suggesting that the sediments are composed of biogenic iron oxides. At Volcano 19, an areally extensive, active vent field, the microbial cells appeared to be responsible for the formation of cohesive chimney-like structures of iron oxyhydroxide, 2-3 m in height, whereas at Volcano 1, an older vent field, no chimney-like structures were apparent. Iron reduction of the sediment material (i.e. BIOS) by Shewanella putrefaciens CN32 was measured, in vitro, as the ratio of [total Fe(II)]:[total Fe]. From this parameter, reduction rates were calculated for Volcano 1 BIOS (0.0521 day(-1)), Volcano 19 BIOS (0.0473 day(-1)), and hydrous ferric oxide, a synthetic two-line ferrihydrite (0.0224 day(-1)). Sediments from both BIOS sites were more easily reduced than synthetic ferrihydrite, which suggests that the decrease in effective surface area of the minerals within the sediments (due to the presence of the organic component) does not inhibit subsequent microbial reduction. These results indicate that natural, marine BIOS are easily reduced in the presence of dissimilatory iron-reducing bacteria, and that the

  8. Putative contact ketoconazole shampoo-triggered pemphigus foliaceus in a dog.

    Science.gov (United States)

    Sung, Hyun-Jeong; Yoon, In-Hwa; Kim, Jung-Hyun

    2017-09-01

    A 10-year-old spayed female cocker spaniel dog was referred for an evaluation of acute-onset generalized pustular cutaneous lesions following application of ketoconazole shampoo. Cytologic and histopathologic examinations of the lesions revealed intra-epidermal pustules with predominantly neutrophils and acantholytic cells. This is the first description of putative contact ketoconazole shampoo-triggered pemphigus foliaceus in a dog.

  9. Differential expressions of putative genes in various floral organs of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-06-03

    Jun 3, 2009 ... Full Length Research Paper. Differential expressions of putative genes in various floral organs of the Pigeon orchid (Dendrobium crumenatum) using GeneFishing. Faridah, Q. Z.1, 2, Ng, B. Z.3, Raha, A. R.4, Umi, K. A. B.5 and Khosravi, A. R.2*. 1Department of Biology, Faculty Science, University Putra ...

  10. A genetic link between epigenetic repressor AS1-AS2 and a putative small subunit processome in leaf polarity establishment of Arabidopsis

    Directory of Open Access Journals (Sweden)

    Yoko Matsumura

    2016-07-01

    Full Text Available Although the DEAD-box RNA helicase family is ubiquitous in eukaryotes, its developmental role remains unelucidated. Here, we report that cooperative action between the Arabidopsis nucleolar protein RH10, an ortholog of human DEAD-box RNA helicase DDX47, and the epigenetic repressor complex of ASYMMETRIC-LEAVES1 (AS1 and AS2 (AS1-AS2 is critical to repress abaxial (ventral genes ETT/ARF3 and ARF4, which leads to adaxial (dorsal development in leaf primordia at shoot apices. Double mutations of rh10-1 and as2 (or as1 synergistically up-regulated the abaxial genes, which generated abaxialized filamentous leaves with loss of the adaxial domain. DDX47 is part of the small subunit processome (SSUP that mediates rRNA biogenesis. In rh10-1 we found various defects in SSUP-related events, such as: accumulation of 35S/33S rRNA precursors; reduction in the 18S/25S ratio; and nucleolar hypertrophy. Double mutants of as2 with mutations of genes that encode other candidate SSUP-related components such as nucleolin and putative rRNA methyltransferase exhibited similar synergistic defects caused by up-regulation of ETT/ARF3 and ARF4. These results suggest a tight link between putative SSUP and AS1-AS2 in repression of the abaxial-determining genes for cell fate decisions for adaxial development.

  11. Inhibitory Synaptic Plasticity - Spike timing dependence and putative network function.

    Directory of Open Access Journals (Sweden)

    Tim P Vogels

    2013-07-01

    Full Text Available While the plasticity of excitatory synaptic connections in the brain has been widely studied, the plasticity of inhibitory connections is much less understood. Here, we present recent experimental and theoretical □ndings concerning the rules of spike timing-dependent inhibitory plasticity and their putative network function. This is a summary of a workshop at the COSYNE conference 2012.

  12. Cloning and sequence analysis of putative type II fatty acid synthase ...

    Indian Academy of Sciences (India)

    Prakash

    Cloning and sequence analysis of putative type II fatty acid synthase genes from Arachis hypogaea L. ... acyl carrier protein (ACP), malonyl-CoA:ACP transacylase, β-ketoacyl-ACP .... Helix II plays a dominant role in the interaction ... main distinguishing features of plant ACPs in plastids and ..... synthase component; J. Biol.

  13. Putative golden proportions as predictors of facial esthetics in adolescents.

    Science.gov (United States)

    Kiekens, Rosemie M A; Kuijpers-Jagtman, Anne Marie; van 't Hof, Martin A; van 't Hof, Bep E; Maltha, Jaap C

    2008-10-01

    In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. Seventy-six adult laypeople evaluated sets of photographs of 64 adolescents on a visual analog scale (VAS) from 0 to 100. The facial esthetic value of each subject was calculated as a mean VAS score. Three observers recorded the position of 13 facial landmarks included in 19 putative golden proportions, based on the golden proportions as defined by Ricketts. The proportions and each proportion's deviation from the golden target (1.618) were calculated. This deviation was then related to the VAS scores. Only 4 of the 19 proportions had a significant negative correlation with the VAS scores, indicating that beautiful faces showed less deviation from the golden standard than less beautiful faces. Together, these variables explained only 16% of the variance. Few golden proportions have a significant relationship with facial esthetics in adolescents. The explained variance of these variables is too small to be of clinical importance.

  14. Memory-guided sensory comparisons in the prefrontal cortex: contribution of putative pyramidal cells and interneurons.

    Science.gov (United States)

    Hussar, Cory R; Pasternak, Tatiana

    2012-02-22

    Comparing two stimuli that occur at different times demands the coordination of bottom-up and top-down processes. It has been hypothesized that the dorsolateral prefrontal (PFC) cortex, the likely source of top-down cortical influences, plays a key role in such tasks, contributing to both maintenance and sensory comparisons. We examined this hypothesis by recording from the PFC of monkeys comparing directions of two moving stimuli, S1 and S2, separated by a memory delay. We determined the contribution of the two principal cell types to these processes by classifying neurons into broad-spiking (BS) putative pyramidal cells and narrow-spiking (NS) putative local interneurons. During the delay, BS cells were more likely to exhibit anticipatory modulation and represent the remembered direction. While this representation was transient, appearing at different times in different neurons, it weakened when direction was not task relevant, suggesting its utility. During S2, both putative cell types showed comparison-related activity modulations. These modulations were of two types, each carried by different neurons, which either preferred trials with stimuli moving in the same direction or trials with stimuli of different directions. These comparison effects were strongly correlated with choice, suggesting their role in circuitry underlying decision making. These results provide the first demonstration of distinct contributions made by principal cell types to memory-guided perceptual decisions. During sensory stimulation both cell types represent behaviorally relevant stimulus features contributing to comparison and decision-related activity. However in the absence of sensory stimulation, putative pyramidal cells dominated, carrying information about the elapsed time and the preceding direction.

  15. Crystallization and preliminary crystallographic analysis of a putative glucokinase/hexokinase from Thermus thermophilus

    International Nuclear Information System (INIS)

    Nakamura, Tsutomu; Kashima, Yasuhiro; Mine, Shouhei; Oku, Takashi; Uegaki, Koichi

    2011-01-01

    In this study, a putative glucokinase/hexokinase from T. thermophilus was purified and crystallized. Diffraction data were collected and processed to 2.02 Å resolution. Glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. The open reading frame TTHA0299 of the extreme thermophile Thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. In this study, the glucokinase/hexokinase from T. thermophilus was purified and crystallized using polyethylene glycol 8000 as a precipitant. Diffraction data were collected and processed to 2.02 Å resolution. The crystal belonged to space group P2 1 , with unit-cell parameters a = 70.93, b = 138.14, c = 75.16 Å, β = 95.41°

  16. Evaluation of surveillance of dengue fever cases in the public health centre of Putat Jaya based on attribute surveillance

    Directory of Open Access Journals (Sweden)

    Zumaroh Zumaroh

    2015-01-01

    Full Text Available Dengue Hemorrhagic Fever (DHF is a public health problem in the village of Putat Jaya which is an endemic area. Surveilans activity in DHF control program is the most important activity in controlling and monitoring disease progression. The program is expected to achieve incidence rate 55/100.000 population. This study aimed to evaluate the implementation of case surveilans in health centre of putat jaya based on attribute surveillance. Attribute surveillance is an indicator that describes the characteristics of the surveillance system. This research was an evaluation research with descriptive study design. As informants were clinic staff who deal specifically with cases of dengue hemorrhagic fever and laboratory workers. The techniques of data collection by interviews and document study. The variables of this study were simplicity, flexibility, acceptability, sensitivity, positive predictive value, representativeness, timeliness, data quality and data stability. It could be seen from Incidence Rate in 2013 has reached 133/100.00 population. The activity of surveilance in the village of Putat Jaya reviewed from disease contol program management was not succeed into decrease incidence rate of DHF. Therefore, dengue control programs in health centers Putat Jaya need to do cross-sector cooperation and cross-program cooperation, strengthening the case reporting system by way increasing in the utilization of information and communication technology electromedia. Keywords: case surveillance, dengue hemorrhagic fever, evaluation, attribute surveillance, Putat Jaya

  17. Genome sequence of the organohalide-respiring Dehalogenimonas alkenigignens type strain (IP3-3(T)).

    Science.gov (United States)

    Key, Trent A; Richmond, Dray P; Bowman, Kimberly S; Cho, Yong-Joon; Chun, Jongsik; da Costa, Milton S; Rainey, Fred A; Moe, William M

    2016-01-01

    Dehalogenimonas alkenigignens IP3-3(T) is a strictly anaerobic, mesophilic, Gram negative staining bacterium that grows by organohalide respiration, coupling the oxidation of H2 to the reductive dehalogenation of polychlorinated alkanes. Growth has not been observed with any non-polyhalogenated alkane electron acceptors. Here we describe the features of strain IP3-3(T) together with genome sequence information and its annotation. The 1,849,792 bp high-quality-draft genome contains 1936 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small unit rRNA (16S) locus. The genome contains 29 predicted reductive dehalogenase genes, a large majority of which lack cognate genes encoding membrane anchoring proteins.

  18. Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

    Science.gov (United States)

    Lawo, N C; Griesser, M; Forneck, A

    Grape phylloxera ( Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir , we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C ( V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

  19. Distribution of putative virulence genes and antimicrobial drug resistance in Vibrio harveyi

    Digital Repository Service at National Institute of Oceanography (India)

    Parvathi, A.; Mendez, D.; Anto, C.

    zonula occludens toxin (Zot) and a hemolysin-coregulated protein gene (hcp) by polymerase chain reaction (PCR). Of the four putative reversible toxin genes, vhh-1 was detected in 31% of the isolates, vhh-2 in 46%, vhh-3 in 23% and vhh-4 was detected in 27...

  20. Cloning and characterization of prunus serotina AGAMOUS, a putative flower homeotic gene

    Science.gov (United States)

    Xiaomei Liu; Joseph Anderson; Paula Pijut

    2010-01-01

    Members of the AGAMOUS subfamily of MADS-box transcription factors play an important role in regulating the development of reproductive organs in flowering plants. To help understand the mechanism of floral development in black cherry (Prunus serotina), PsAG (a putative flower homeotic identity gene) was isolated...

  1. Transcriptomic analysis on the formation of the viable putative non-culturable state of beer-spoilage Lactobacillus acetotolerans.

    Science.gov (United States)

    Liu, Junyan; Deng, Yang; Peters, Brian M; Li, Lin; Li, Bing; Chen, Lequn; Xu, Zhenbo; Shirtliff, Mark E

    2016-11-07

    Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria regardless of beer type, and thus pose significant problems for the brewery industry. The aim of this study was to investigate the genetic mechanisms involved in the ability of the hard-to-culture beer-spoilage bacterium Lactobacillus acetotolerans to enter into the viable putative non-culturable (VPNC) state. A genome-wide transcriptional analysis of beer-spoilage L. acetotolerans strains BM-LA14526, BM-LA14527, and BM-LA14528 under normal, mid-term and VPNC states were performed using RNA-sequencing (RNA-seq) and further bioinformatics analyses. GO function, COG category, and KEGG pathway enrichment analysis were conducted to investigate functional and related metabolic pathways of the differentially expressed genes. Functional and pathway enrichment analysis indicated that heightened stress response and reduction in genes associated with transport, metabolic process, and enzyme activity might play important roles in the formation of the VPNC state. This is the first transcriptomic analysis on the formation of the VPNC state of beer spoilage L. acetotolerans.

  2. Putative neuroprotective agents in neuropsychiatric disorders.

    Science.gov (United States)

    Dodd, Seetal; Maes, Michael; Anderson, George; Dean, Olivia M; Moylan, Steven; Berk, Michael

    2013-04-05

    In many individuals with major neuropsychiatric disorders including depression, bipolar disorder and schizophrenia, their disease characteristics are consistent with a neuroprogressive illness. This includes progressive structural brain changes, cognitive and functional decline, poorer treatment response and an increasing vulnerability to relapse with chronicity. The underlying molecular mechanisms of neuroprogression are thought to include neurotrophins and regulation of neurogenesis and apoptosis, neurotransmitters, inflammatory, oxidative and nitrosative stress, mitochondrial dysfunction, cortisol and the hypothalamic-pituitary-adrenal axis, and epigenetic influences. Knowledge of the involvement of each of these pathways implies that specific agents that act on some or multiple of these pathways may thus block this cascade and have neuroprotective properties. This paper reviews the potential of the most promising of these agents, including lithium and other known psychotropics, aspirin, minocycline, statins, N-acetylcysteine, leptin and melatonin. These agents are putative neuroprotective agents for schizophrenia and mood disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Distribution and innervation of putative peripheral arterial chemoreceptors in the red-eared slider (Trachemys scripta elegans).

    Science.gov (United States)

    Reyes, Catalina; Fong, Angelina Y; Milsom, William K

    2015-06-15

    Peripheral arterial chemoreceptors have been isolated to the common carotid artery, aorta, and pulmonary artery of turtles. However, the putative neurotransmitters associated with these chemoreceptors have not yet been described. The goal of the present study was to determine the neurochemical content, innervations, and distribution of putative oxygen-sensing cells in the central vasculature of turtles and to derive homologies with peripheral arterial chemoreceptors of other vertebrates. We used tract tracing together with immunohistochemical markers for cholinergic cells (vesicular acetylcholine transporter [VAChT]), tyrosine hydroxylase (TH; the rate-limiting enzyme in catecholamine synthesis), and serotonin (5HT) to identify putative oxygen-sensing cells and to determine their anatomical relation to branches of the vagus nerve (Xth cranial nerve). We found potential oxygen-sensing cells in all three chemosensory areas innervated by branches of the Xth cranial nerve. Cells containing either 5HT or VAChT were found in all three sites. The morphology and size of these cells resemble glomus cells found in amphibians, mammals, tortoises, and lizards. Furthermore, we found populations of cholinergic cells located at the base of the aorta and pulmonary artery that are likely involved in efferent regulation of vessel resistance. Catecholamine-containing cells were not found in any of the putative chemosensitive areas. The presence of 5HT- and VAChT-immunoreactive cells in segments of the common carotid artery, aorta, and pulmonary artery appears to reflect a transition between cells containing the major neurotransmitters seen in fish (5HT) and mammals (ACh and adenosine). © 2015 Wiley Periodicals, Inc.

  4. Draft genome sequence of Streptomyces coelicoflavus ZG0656 reveals the putative biosynthetic gene cluster of acarviostatin family α-amylase inhibitors.

    Science.gov (United States)

    Guo, X; Geng, P; Bai, F; Bai, G; Sun, T; Li, X; Shi, L; Zhong, Q

    2012-08-01

    The aims of this study are to obtain the draft genome sequence of Streptomyces coelicoflavus ZG0656, which produces novel acarviostatin family α-amylase inhibitors, and then to reveal the putative acarviostatin-related gene cluster and the biosynthetic pathway. The draft genome sequence of S. coelicoflavus ZG0656 was generated using a shotgun approach employing a combination of 454 and Solexa sequencing technologies. Genome analysis revealed a putative gene cluster for acarviostatin biosynthesis, termed sct-cluster. The cluster contains 13 acarviostatin synthetic genes, six transporter genes, four starch degrading or transglycosylation enzyme genes and two regulator genes. On the basis of bioinformatic analysis, we proposed a putative biosynthetic pathway of acarviostatins. The intracellular steps produce a structural core, acarviostatin I00-7-P, and the extracellular assemblies lead to diverse acarviostatin end products. The draft genome sequence of S. coelicoflavus ZG0656 revealed the putative biosynthetic gene cluster of acarviostatins and a putative pathway of acarviostatin production. To our knowledge, S. coelicoflavus ZG0656 is the first strain in this species for which a genome sequence has been reported. The analysis of sct-cluster provided important insights into the biosynthesis of acarviostatins. This work will be a platform for producing novel variants and yield improvement. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

  5. Hypothesis: Gene-rich plastid genomes in red algae may be an outcome of nuclear genome reduction.

    Science.gov (United States)

    Qiu, Huan; Lee, Jun Mo; Yoon, Hwan Su; Bhattacharya, Debashish

    2017-06-01

    Red algae (Rhodophyta) putatively diverged from the eukaryote tree of life >1.2 billion years ago and are the source of plastids in the ecologically important diatoms, haptophytes, and dinoflagellates. In general, red algae contain the largest plastid gene inventory among all such organelles derived from primary, secondary, or additional rounds of endosymbiosis. In contrast, their nuclear gene inventory is reduced when compared to their putative sister lineage, the Viridiplantae, and other photosynthetic lineages. The latter is thought to have resulted from a phase of genome reduction that occurred in the stem lineage of Rhodophyta. A recent comparative analysis of a taxonomically broad collection of red algal and Viridiplantae plastid genomes demonstrates that the red algal ancestor encoded ~1.5× more plastid genes than Viridiplantae. This difference is primarily explained by more extensive endosymbiotic gene transfer (EGT) in the stem lineage of Viridiplantae, when compared to red algae. We postulate that limited EGT in Rhodophytes resulted from the countervailing force of ancient, and likely recurrent, nuclear genome reduction. In other words, the propensity for nuclear gene loss led to the retention of red algal plastid genes that would otherwise have undergone intracellular gene transfer to the nucleus. This hypothesis recognizes the primacy of nuclear genome evolution over that of plastids, which have no inherent control of their gene inventory and can change dramatically (e.g., secondarily non-photosynthetic eukaryotes, dinoflagellates) in response to selection acting on the host lineage. © 2017 Phycological Society of America.

  6. Semi-automated literature mining to identify putative biomarkers of disease from multiple biofluids

    Science.gov (United States)

    2014-01-01

    Background Computational methods for mining of biomedical literature can be useful in augmenting manual searches of the literature using keywords for disease-specific biomarker discovery from biofluids. In this work, we develop and apply a semi-automated literature mining method to mine abstracts obtained from PubMed to discover putative biomarkers of breast and lung cancers in specific biofluids. Methodology A positive set of abstracts was defined by the terms ‘breast cancer’ and ‘lung cancer’ in conjunction with 14 separate ‘biofluids’ (bile, blood, breastmilk, cerebrospinal fluid, mucus, plasma, saliva, semen, serum, synovial fluid, stool, sweat, tears, and urine), while a negative set of abstracts was defined by the terms ‘(biofluid) NOT breast cancer’ or ‘(biofluid) NOT lung cancer.’ More than 5.3 million total abstracts were obtained from PubMed and examined for biomarker-disease-biofluid associations (34,296 positive and 2,653,396 negative for breast cancer; 28,355 positive and 2,595,034 negative for lung cancer). Biological entities such as genes and proteins were tagged using ABNER, and processed using Python scripts to produce a list of putative biomarkers. Z-scores were calculated, ranked, and used to determine significance of putative biomarkers found. Manual verification of relevant abstracts was performed to assess our method’s performance. Results Biofluid-specific markers were identified from the literature, assigned relevance scores based on frequency of occurrence, and validated using known biomarker lists and/or databases for lung and breast cancer [NCBI’s On-line Mendelian Inheritance in Man (OMIM), Cancer Gene annotation server for cancer genomics (CAGE), NCBI’s Genes & Disease, NCI’s Early Detection Research Network (EDRN), and others]. The specificity of each marker for a given biofluid was calculated, and the performance of our semi-automated literature mining method assessed for breast and lung cancer

  7. DETERMINATION OF ROCURONIUM AND ITS PUTATIVE METABOLITES IN BODY-FLUIDS AND TISSUE-HOMOGENATES

    NARCIS (Netherlands)

    KLEEF, UW; PROOST, JH; ROGGEVELD, J

    1993-01-01

    A sensitive and selective HPLC method was developed for the quantification of the neuromuscular blocking agent rocuronium and its putative metabolites (the 17-desacetyl derivative and the N-desallyl derivative of rocuronium) in plasma, urine, bile, tissue homogenates and stoma fluid. Samples were

  8. Isolation and characterization of two mitoviruses and a putative alphapartitivirus from Fusarium spp.

    Science.gov (United States)

    Osaki, Hideki; Sasaki, Atsuko; Nomiyama, Koji; Sekiguchi, Hiroyuki; Tomioka, Keisuke; Takehara, Toshiaki

    2015-06-01

    The filamentous fungus Fusarium spp. includes several important plant pathogens. We attempted to reveal presence of double-stranded (ds) RNAs in the genus. Thirty-seven Fusarium spp. at the MAFF collection were analyzed. In the strains of Fusarium coeruleum, Fusarium globosum and Fusarium solani f. sp. pisi, single dsRNA bands were detected. The strains of F. coeruleum and F. solani f. sp. pisi cause potato dry rot and mulberry twig blight, respectively. Sequence analyses revealed that dsRNAs in F. coeruleum and F. globosum consisted of 2423 and 2414 bp, respectively. Using the fungal mitochondrial translation table, the positive strands of these cDNAs were found to contain single open reading frames with the potential to encode a protein of putative 757 and 717 amino acids (molecular mass 88.5 and 84.0 kDa, respectively), similar to RNA-dependent RNA polymerases of members of the genus Mitovirus. These dsRNAs in F. coeruleum and F. globosum were assigned to the genus Mitovirus (family Narnaviridae), and these two mitoviruses were designated as Fusarium coeruleum mitovirus 1 and Fusarium globosum mitovirus 1. On the other hand, a positive strand of cDNA (1950 bp) from dsRNA in F. solani f. sp. pisi contained an ORF potentially encoding a putative RdRp of 608 amino acids (72.0 kDa). The putative RdRp was shown to be related to those of members of the genus of Alphapartitivirus (family Partitiviridae). We coined the name Fusarium solani partitivirus 2 for dsRNA in F. solani f. sp. pisi.

  9. Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria

    Directory of Open Access Journals (Sweden)

    Cui Hongli

    2012-11-01

    Full Text Available Abstract Background Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS. PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Results Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. Conclusions The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic

  10. Genome-wide analysis of putative peroxiredoxin in unicellular and filamentous cyanobacteria.

    Science.gov (United States)

    Cui, Hongli; Wang, Yipeng; Wang, Yinchu; Qin, Song

    2012-11-16

    Cyanobacteria are photoautotrophic prokaryotes with wide variations in genome sizes and ecological habitats. Peroxiredoxin (PRX) is an important protein that plays essential roles in protecting own cells against reactive oxygen species (ROS). PRXs have been identified from mammals, fungi and higher plants. However, knowledge on cyanobacterial PRXs still remains obscure. With the availability of 37 sequenced cyanobacterial genomes, we performed a comprehensive comparative analysis of PRXs and explored their diversity, distribution, domain structure and evolution. Overall 244 putative prx genes were identified, which were abundant in filamentous diazotrophic cyanobacteria, Acaryochloris marina MBIC 11017, and unicellular cyanobacteria inhabiting freshwater and hot-springs, while poor in all Prochlorococcus and marine Synechococcus strains. Among these putative genes, 25 open reading frames (ORFs) encoding hypothetical proteins were identified as prx gene family members and the others were already annotated as prx genes. All 244 putative PRXs were classified into five major subfamilies (1-Cys, 2-Cys, BCP, PRX5_like, and PRX-like) according to their domain structures. The catalytic motifs of the cyanobacterial PRXs were similar to those of eukaryotic PRXs and highly conserved in all but the PRX-like subfamily. Classical motif (CXXC) of thioredoxin was detected in protein sequences from the PRX-like subfamily. Phylogenetic tree constructed of catalytic domains coincided well with the domain structures of PRXs and the phylogenies based on 16s rRNA. The distribution of genes encoding PRXs in different unicellular and filamentous cyanobacteria especially those sub-families like PRX-like or 1-Cys PRX correlate with the genome size, eco-physiology, and physiological properties of the organisms. Cyanobacterial and eukaryotic PRXs share similar conserved motifs, indicating that cyanobacteria adopt similar catalytic mechanisms as eukaryotes. All cyanobacterial PRX proteins

  11. Homoacetogenesis in Deep-Sea Chloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in Terrestrial Dehalococcoidetes.

    Science.gov (United States)

    Sewell, Holly L; Kaster, Anne-Kristin; Spormann, Alfred M

    2017-12-19

    The deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylum Chloroflexi In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in such Chloroflexi 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum I Chloroflexi which were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, a Rhodobacter nitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marine Chloroflexi We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylum Chloroflexi (DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrial Dehalococcoides/Dehalogenimonas spp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity in Dehalococcoides/Dehalogenimonas spp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrial Dehalococcoides/Dehalogenimonas spp. and of reductive

  12. Identification of EhTIF-IA: The putative E. histolytica orthologue of the ...

    Indian Academy of Sciences (India)

    2016-02-04

    Feb 4, 2016 ... We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within ..... a putative EhTIF-IA with e-value (3e−25). Comparison of .... some biogenesis is correlated with altered rates of rDNA transcription ..... ylation by CK2 facilitates rDNA transcription by promoting dissociation of ...

  13. Discovery of Putative Herbicide Resistance Genes and Its Regulatory Network in Chickpea Using Transcriptome Sequencing

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    Mir A. Iquebal

    2017-06-01

    Full Text Available Background: Chickpea (Cicer arietinum L. contributes 75% of total pulse production. Being cheaper than animal protein, makes it important in dietary requirement of developing countries. Weed not only competes with chickpea resulting into drastic yield reduction but also creates problem of harboring fungi, bacterial diseases and insect pests. Chemical approach having new herbicide discovery has constraint of limited lead molecule options, statutory regulations and environmental clearance. Through genetic approach, transgenic herbicide tolerant crop has given successful result but led to serious concern over ecological safety thus non-transgenic approach like marker assisted selection is desirable. Since large variability in tolerance limit of herbicide already exists in chickpea varieties, thus the genes offering herbicide tolerance can be introgressed in variety improvement programme. Transcriptome studies can discover such associated key genes with herbicide tolerance in chickpea.Results: This is first transcriptomic studies of chickpea or even any legume crop using two herbicide susceptible and tolerant genotypes exposed to imidazoline (Imazethapyr. Approximately 90 million paired-end reads generated from four samples were processed and assembled into 30,803 contigs using reference based assembly. We report 6,310 differentially expressed genes (DEGs, of which 3,037 were regulated by 980 miRNAs, 1,528 transcription factors associated with 897 DEGs, 47 Hub proteins, 3,540 putative Simple Sequence Repeat-Functional Domain Marker (SSR-FDM, 13,778 genic Single Nucleotide Polymorphism (SNP putative markers and 1,174 Indels. Randomly selected 20 DEGs were validated using qPCR. Pathway analysis suggested that xenobiotic degradation related gene, glutathione S-transferase (GST were only up-regulated in presence of herbicide. Down-regulation of DNA replication genes and up-regulation of abscisic acid pathway genes were observed. Study further reveals

  14. Putative midkine family protein up-regulation in Patella caerulea (Mollusca, Gastropoda) exposed to sublethal concentrations of cadmium

    International Nuclear Information System (INIS)

    Vanucci, Silvana; Minerdi, Daniela; Kadomatsu, Kenji; Mengoni, Alessio; Bazzicalupo, Marco

    2005-01-01

    A cDNA sequence of a putative midkine (MK) family protein was identified and characterised in the mollusc Patella caerulea. The midkine family consists of two members, midkine and pleiotrophin (PTN), and it is one of the recently discovered cytokines. Our results show that this putative midkine protein is up-regulated in specimens of P. caerulea exposed to sublethal cadmium concentrations (i.e. 0.5 and 1 mg l -1 Cd) over a 10-day exposure period. Semiquantitative RT-PCR and quantitative Real time RT-PCR estimations indicate elevated expression of midkine mRNA in exposed specimens compared to controls. Moreover, RT-PCR Real time values were higher in the viscera (here defined as the part of the soft tissue including digestive gland plus gills) than in the foot (i.e. foot plus head plus heart) of the limpets. At present, information on the functional signalling significance of the midkine family proteins suggests that the up-regulation of P. caerulea putative midkine family protein is a distress signal likely with informative value on health status of the organism and with potential prognostic capability

  15. Total synthesis of the putative structure of the novel triquinane natural product isocapnellenone

    OpenAIRE

    Mehta, Goverdhan; Murthy, Sai Krishna A; Umarye, Jayant D

    2002-01-01

    A total synthesis of the ‘putative structure’ 7, attributed to the novel triquinane sesquiterpene isolated recently from two Buddelia species has been accomplished. The spectral data for 7 is a complete mismatch with those reported for the natural product and warrants a revision of the assigned structure.

  16. Enhanced degradation of haloacid by heterologous expression in related Burkholderia species.

    Science.gov (United States)

    Su, Xianbin; Deng, Liyu; Kong, Ka Fai; Tsang, Jimmy S H

    2013-10-01

    Haloacids are environmental pollutant and can be transformed to non-toxic alkanoic acids by microbial dehalogenase. Bacterium Burkholderia species MBA4 was enriched from soil for its ability to bioremediate haloacids such as mono-chloroacetate (MCA), mono-bromoacetate (MBA), 2-mono-chloropropionate, and 2-mono-bromopropionate. MBA4 produces an inducible dehalogenase Deh4a that catalyzes the dehalogenation process. The growth of MBA4 on haloacid also relies on the presence of a haloacid-uptake system. Similar dehalogenase genes can be found in the genome of many related species. However, wildtype Burkholderia caribensis MWAP64, Burkholderia phymatum STM815, and Burkholderia xenovorans LB400 were not able to grow on MCA. When a plasmid containing the regulatory and structural gene of Deh4a was transformed to these species, they were able to grow on haloacid. The specific enzyme activities in these recombinants ranges from 2- to 30-fold that of MBA4 in similar condition. Reverse transcription-quantitative real-time PCR showed that the relative transcript levels in these recombinant strains ranges from 9 to over 1,600 times that of MBA4 in similar condition. A recombinant has produced nearly five times of dehalogenase that MBA4 could ever achieve. While the expressions of Deh4a were more relaxed in these phylogenetically related species, an MCA-uptake activity was found to be inducible. These metabolically engineered strains are better degraders than the haloacid-enriched MBA4. Copyright © 2013 Wiley Periodicals, Inc.

  17. KLONING GEN PUTATIVE CLEAVAGE PROTEIN 1 (PCP-1 PADA UDANG VANAME (Litopenaeus vannamei YANG TERSERANG INFECTIOUS MYONECROSIS VIRUS

    Directory of Open Access Journals (Sweden)

    Hessy Novita

    2016-12-01

    Full Text Available Penanggulangan penyakit ikan dapat dilakukan dengan cara meningkatkan kekebalan tubuh ikan melalui program vaksinasi. Namun vaksinasi tidak tepat untuk udang, karena udang tidak mempunyai immunological memory seperti ikan. Oleh karena itu, perlindungan udang terhadap serangan penyakit viral dengan menggunakan RNA interference (RNAi. Teknologi RNAi digunakan untuk menghalangi (interfere proses replikasi infectious myonecrosis virus (IMNV pada udang vaname dengan cara menon-aktifkan gen putative cleavage protein 1 (PCP-1, yang berfungsi dalam pembentukan capsid dan proses transkripsi RNA IMNV. Penelitian ini bertujuan untuk melakukan kloning gen putative cleavage protein 1 dalam rangka perakitan teknologi RNAi untuk pengendalian penyakit IMNV pada udang vaname. Tahapan penelitian meliputi koleksi sampel, isolasi RNA, sintesis cDNA, amplifikasi PCR, purifikasi DNA, transformasi, isolasi plasmid, serta sekuensing dan analisis data. Hasil isolasi plasmid cDNA PCP-1 memperlihatkan semua koloni bakteri terseleksi ternyata membawa plasmid hasil insersi DNA gen PCP–1, hasil sekuen dengan nilai homologinya mencapai 100% dan 99% yang dibandingkan dengan sekuen di Genebank. Hasil penelitian menunjukkan bahwa kloning gen putative cleavage protein 1 (PCP-1 dari udang vaname yang terserang Infectious Myonecrosis Virus berhasil dikloning yang nantinya digunakan untuk perakitan RNAi. The prevention of fish diseases can be done by increasing of the fish immune through vaccination programs. However, the vaccination can not be done for the shrimp,due to the absence of  immunological memory. Therefore, the protection of shrimp against viral diseases was done by using of RNA interference (RNAi. RNAi technology is used to interfere infectious myonecrosis virus (IMNV replication process on white shrimp by disabling of putative cleavage protein 1 (PCP-1gene, which functions in capsid formation and RNA transcription process. The study was conducted to perform putative

  18. Are MAO-A deficiency states in the general population and in putative high-risk populations highly uncommon?

    Science.gov (United States)

    Murphy, D L; Sims, K; Eisenhofer, G; Greenberg, B D; George, T; Berlin, F; Zametkin, A; Ernst, M; Breakefield, X O

    1998-01-01

    Lack of monoamine oxidase A (MAO-A) due to either Xp chromosomal deletions or alterations in the coding sequence of the gene for this enzyme are associated with marked changes in monoamine metabolism and appear to be associated with variable cognitive deficits and behavioral changes in humans and in transgenic mice. In mice, some of the most marked behavioral changes are ameliorated by pharmacologically-induced reductions in serotonin synthesis during early development, raising the question of possible therapeutic interventions in humans with MAO deficiency states. At the present time, only one multi-generational family and a few other individuals with marked MAO-A deficiency states have been identified and studied in detail. Although MAO deficiency states associated with Xp chromosomal deletions were identified by distinct symptoms (including blindness in infancy) produced by the contiguous Norrie disease gene, the primarily behavioral phenotype of individuals with the MAO mutation is less obvious. This paper reports a sequential research design and preliminary results from screening several hundred volunteers in the general population and from putative high-risk groups for possible MAO deficiency states. These preliminary results suggest that marked MAO deficiency states are very rare.

  19. Investigation of paternity establishing without the putative father using hypervariable DNA probes.

    Science.gov (United States)

    Yokoi, T; Odaira, T; Nata, M; Sagisaka, K

    1990-09-01

    Seven kinds of DNA probes which recognize hypervariable loci were applied for paternity test. The putative father was decreased and unavailable for the test. The two legitimate children and their mother (the deceased's wife) and the four illegitimate children and their mother (the deceased's kept mistress) were available for analysis. Paternity index of four illegitimate child was investigated. Allelic frequencies and their confidence intervals among unrelated Japanese individuals were previously reported from our laboratory, and co-dominant segregation of the polymorphism was confirmed in family studies. Cumulative paternity indices of four illegitimate children from 16 kinds of standard blood group markers were 165, 42, 0.09, and 36, respectively. On the other hand, cumulative paternity indices from 7 kinds of DNA probes are 2,363, 4,685, 57,678, and 54,994, respectively, which are 14, 113, 640, 864, and 1,509 times higher than that from standard blood group markers. The DNA analyses gave nearly conclusive evidence that the putative father was the biological father of the children. Especially, the paternity relation of the third illegitimate child could not be established without the DNA analyses. Accordingly, DNA polymorphism is considered to be informative enough for paternity test.

  20. The solution structure of ChaB, a putative membrane ion antiporter regulator from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Iannuzzi Pietro

    2004-08-01

    Full Text Available Abstract Background ChaB is a putative regulator of ChaA, a Na+/H+ antiporter that also has Ca+/H+ activity in E. coli. ChaB contains a conserved 60-residue region of unknown function found in other bacteria, archaeabacteria and a series of baculoviral proteins. As part of a structural genomics project, the structure of ChaB was elucidated by NMR spectroscopy. Results The structure of ChaB is composed of 3 α-helices and a small sheet that pack tightly to form a fold that is found in the cyclin-box family of proteins. Conclusion ChaB is distinguished from its putative DNA binding sequence homologues by a highly charged flexible loop region that has weak affinity to Mg2+ and Ca2+ divalent metal ions.

  1. Computational Exploration of Putative LuxR Solos in Archaea and Their Functional Implications in Quorum Sensing

    Science.gov (United States)

    Rajput, Akanksha; Kumar, Manoj

    2017-01-01

    LuxR solos are unexplored in Archaea, despite their vital role in the bacterial regulatory network. They assist bacteria in perceiving acyl homoserine lactones (AHLs) and/or non-AHLs signaling molecules for establishing intraspecies, interspecies, and interkingdom communication. In this study, we explored the potential LuxR solos of Archaea from InterPro v62.0 meta-database employing taxonomic, probable function, distribution, and evolutionary aspects to decipher their role in quorum sensing (QS). Our bioinformatics analyses showed that putative LuxR solos of Archaea shared few conserved domains with bacterial LuxR despite having less similarity within proteins. Functional characterization revealed their ability to bind various AHLs and/or non-AHLs signaling molecules that involve in QS cascades alike bacteria. Further, the phylogenetic study indicates that Archaeal LuxR solos (with less substitution per site) evolved divergently from bacteria and share distant homology along with instances of horizontal gene transfer. Moreover, Archaea possessing putative LuxR solos, exhibit the correlation between taxonomy and ecological niche despite being the inhabitant of diverse habitats like halophilic, thermophilic, barophilic, methanogenic, and chemolithotrophic. Therefore, this study would shed light in deciphering the role of the putative LuxR solos of Archaea to adapt varied habitats via multilevel communication with other organisms using QS. PMID:28515720

  2. Computational Exploration of Putative LuxR Solos in Archaea and Their Functional Implications in Quorum Sensing

    Directory of Open Access Journals (Sweden)

    Akanksha Rajput

    2017-05-01

    Full Text Available LuxR solos are unexplored in Archaea, despite their vital role in the bacterial regulatory network. They assist bacteria in perceiving acyl homoserine lactones (AHLs and/or non-AHLs signaling molecules for establishing intraspecies, interspecies, and interkingdom communication. In this study, we explored the potential LuxR solos of Archaea from InterPro v62.0 meta-database employing taxonomic, probable function, distribution, and evolutionary aspects to decipher their role in quorum sensing (QS. Our bioinformatics analyses showed that putative LuxR solos of Archaea shared few conserved domains with bacterial LuxR despite having less similarity within proteins. Functional characterization revealed their ability to bind various AHLs and/or non-AHLs signaling molecules that involve in QS cascades alike bacteria. Further, the phylogenetic study indicates that Archaeal LuxR solos (with less substitution per site evolved divergently from bacteria and share distant homology along with instances of horizontal gene transfer. Moreover, Archaea possessing putative LuxR solos, exhibit the correlation between taxonomy and ecological niche despite being the inhabitant of diverse habitats like halophilic, thermophilic, barophilic, methanogenic, and chemolithotrophic. Therefore, this study would shed light in deciphering the role of the putative LuxR solos of Archaea to adapt varied habitats via multilevel communication with other organisms using QS.

  3. Crystal Structure of a Putative HTH-Type Transcriptional Regulator yxaF from Bacillus subtilis

    International Nuclear Information System (INIS)

    Seetharaman, J.; Kumaran, D.; Bonanno, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    The New York Structural GenomiX Research Consortium (NYSGXRC) has selected the protein coded by yxaF gene from Bacillus subtilis as a target for structure determination. The yxaF protein has 191 residues with a molecular mass of 21 kDa and had no sequence homology to any structure in the Protein Data Bank (PDB) at the time of target selection. We aimed to elucidate the three-dimensional structure for the putative protein yxaF to better understand the relationship between protein sequence, structure, and function. This protein is annotated as a putative helix-turn-helix (HTH) type transcriptional regulator. Many transcriptional regulators like TetR and QacR use a structurally well-defined DNA-binding HTH motif to recognize the target DNA sequences. DNA-HTH motif interactions have been extensively studied. As the HTH motif is structurally conserved in many regulatory proteins, these DNA-protein complexes show some similarity in DNA recognition patterns. Many such regulatory proteins have a ligand-binding domain in addition to the DNA-binding domain. Structural studies on ligand-binding regulatory proteins provide a wealth of information on ligand-, and possibly drug-, binding mechanisms. Understanding the ligand-binding mechanism may help overcome problems with drug resistance, which represent increasing challenges in medicine. The protein encoded by yxaF, hereafter called T1414, shows fold similar to QacR repressor and TetR/CamR repressor and possesses putative DNA and ligand-binding domains. Here, we report the crystal structure of T1414 and compare it with structurally similar drug and DNA-binding proteins

  4. Identification of Putative Coffee Rust Mycoparasites via Single-Molecule DNA Sequencing of Infected Pustules.

    Science.gov (United States)

    James, Timothy Y; Marino, John A; Perfecto, Ivette; Vandermeer, John

    2016-01-15

    The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests, despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. In the present study, we characterized fungal communities associated with coffee rust lesions by single-molecule DNA sequencing of fungal rRNA gene bar codes from leaf discs (≈28 mm(2)) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyperdiverse in terms of fungi, with up to 69 operational taxonomic units (putative species) per control disc, and the diversity was only slightly reduced in rust-infected discs, with up to 63 putative species. However, geography had a greater influence on the fungal community than whether the disc was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in the fungal family Cordycipitaceae and the order Tremellales. These data emphasize the complexity of diverse fungi of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  5. Identification of genomic variants putatively targeted by selection during dog domestication.

    Science.gov (United States)

    Cagan, Alex; Blass, Torsten

    2016-01-12

    Dogs [Canis lupus familiaris] were the first animal species to be domesticated and continue to occupy an important place in human societies. Recent studies have begun to reveal when and where dog domestication occurred. While much progress has been made in identifying the genetic basis of phenotypic differences between dog breeds we still know relatively little about the genetic changes underlying the phenotypes that differentiate all dogs from their wild progenitors, wolves [Canis lupus]. In particular, dogs generally show reduced aggression and fear towards humans compared to wolves. Therefore, selection for tameness was likely a necessary prerequisite for dog domestication. With the increasing availability of whole-genome sequence data it is possible to try and directly identify the genetic variants contributing to the phenotypic differences between dogs and wolves. We analyse the largest available database of genome-wide polymorphism data in a global sample of dogs 69 and wolves 7. We perform a scan to identify regions of the genome that are highly differentiated between dogs and wolves. We identify putatively functional genomic variants that are segregating or at high frequency [> = 0.75 Fst] for alternative alleles between dogs and wolves. A biological pathways analysis of the genes containing these variants suggests that there has been selection on the 'adrenaline and noradrenaline biosynthesis pathway', well known for its involvement in the fight-or-flight response. We identify 11 genes with putatively functional variants fixed for alternative alleles between dogs and wolves. The segregating variants in these genes are strong candidates for having been targets of selection during early dog domestication. We present the first genome-wide analysis of the different categories of putatively functional variants that are fixed or segregating at high frequency between a global sampling of dogs and wolves. We find evidence that selection has been strongest

  6. Genome-wide characterization of genetic variants and putative regions under selection in meat and egg-type chicken lines.

    Science.gov (United States)

    Boschiero, Clarissa; Moreira, Gabriel Costa Monteiro; Gheyas, Almas Ara; Godoy, Thaís Fernanda; Gasparin, Gustavo; Mariani, Pilar Drummond Sampaio Corrêa; Paduan, Marcela; Cesar, Aline Silva Mello; Ledur, Mônica Corrêa; Coutinho, Luiz Lehmann

    2018-01-25

    Meat and egg-type chickens have been selected for several generations for different traits. Artificial and natural selection for different phenotypes can change frequency of genetic variants, leaving particular genomic footprints throghtout the genome. Thus, the aims of this study were to sequence 28 chickens from two Brazilian lines (meat and white egg-type) and use this information to characterize genome-wide genetic variations, identify putative regions under selection using Fst method, and find putative pathways under selection. A total of 13.93 million SNPs and 1.36 million INDELs were identified, with more variants detected from the broiler (meat-type) line. Although most were located in non-coding regions, we identified 7255 intolerant non-synonymous SNPs, 512 stopgain/loss SNPs, 1381 frameshift and 1094 non-frameshift INDELs that may alter protein functions. Genes harboring intolerant non-synonymous SNPs affected metabolic pathways related mainly to reproduction and endocrine systems in the white-egg layer line, and lipid metabolism and metabolic diseases in the broiler line. Fst analysis in sliding windows, using SNPs and INDELs separately, identified over 300 putative regions of selection overlapping with more than 250 genes. For the first time in chicken, INDEL variants were considered for selection signature analysis, showing high level of correlation in results between SNP and INDEL data. The putative regions of selection signatures revealed interesting candidate genes and pathways related to important phenotypic traits in chicken, such as lipid metabolism, growth, reproduction, and cardiac development. In this study, Fst method was applied to identify high confidence putative regions under selection, providing novel insights into selection footprints that can help elucidate the functional mechanisms underlying different phenotypic traits relevant to meat and egg-type chicken lines. In addition, we generated a large catalog of line-specific and common

  7. A nuclear magnetic resonance based approach to accurate functional annotation of putative enzymes in the methanogen Methanosarcina acetivorans

    Directory of Open Access Journals (Sweden)

    Nikolau Basil J

    2011-06-01

    Full Text Available Abstract Background Correct annotation of function is essential if one is to take full advantage of the vast amounts of genomic sequence data. The accuracy of sequence-based functional annotations is often variable, particularly if the sequence homology to a known function is low. Indeed recent work has shown that even proteins with very high sequence identity can have different folds and functions, and therefore caution is needed in assigning functions by sequence homology in the absence of experimental validation. Experimental methods are therefore needed to efficiently evaluate annotations in a way that complements current high throughput technologies. Here, we describe the use of nuclear magnetic resonance (NMR-based ligand screening as a tool for testing functional assignments of putative enzymes that may be of variable reliability. Results The target genes for this study are putative enzymes from the methanogenic archaeon Methanosarcina acetivorans (MA that have been selected after manual genome re-annotation and demonstrate detectable in vivo expression at the level of the transcriptome. The experimental approach begins with heterologous E. coli expression and purification of individual MA gene products. An NMR-based ligand screen of the purified protein then identifies possible substrates or products from a library of candidate compounds chosen from the putative pathway and other related pathways. These data are used to determine if the current sequence-based annotation is likely to be correct. For a number of case studies, additional experiments (such as in vivo genetic complementation were performed to determine function so that the reliability of the NMR screen could be independently assessed. Conclusions In all examples studied, the NMR screen was indicative of whether the functional annotation was correct. Thus, the case studies described demonstrate that NMR-based ligand screening is an effective and rapid tool for confirming or

  8. Comparative Single-Cell Genomics of Chloroflexi from the Okinawa Trough Deep-Subsurface Biosphere.

    Science.gov (United States)

    Fullerton, Heather; Moyer, Craig L

    2016-05-15

    Chloroflexi small-subunit (SSU) rRNA gene sequences are frequently recovered from subseafloor environments, but the metabolic potential of the phylum is poorly understood. The phylum Chloroflexi is represented by isolates with diverse metabolic strategies, including anoxic phototrophy, fermentation, and reductive dehalogenation; therefore, function cannot be attributed to these organisms based solely on phylogeny. Single-cell genomics can provide metabolic insights into uncultured organisms, like the deep-subsurface Chloroflexi Nine SSU rRNA gene sequences were identified from single-cell sorts of whole-round core material collected from the Okinawa Trough at Iheya North hydrothermal field as part of Integrated Ocean Drilling Program (IODP) expedition 331 (Deep Hot Biosphere). Previous studies of subsurface Chloroflexi single amplified genomes (SAGs) suggested heterotrophic or lithotrophic metabolisms and provided no evidence for growth by reductive dehalogenation. Our nine Chloroflexi SAGs (seven of which are from the order Anaerolineales) indicate that, in addition to genes for the Wood-Ljungdahl pathway, exogenous carbon sources can be actively transported into cells. At least one subunit for pyruvate ferredoxin oxidoreductase was found in four of the Chloroflexi SAGs. This protein can provide a link between the Wood-Ljungdahl pathway and other carbon anabolic pathways. Finally, one of the seven Anaerolineales SAGs contains a distinct reductive dehalogenase homologous (rdhA) gene. Through the use of single amplified genomes (SAGs), we have extended the metabolic potential of an understudied group of subsurface microbes, the Chloroflexi These microbes are frequently detected in the subsurface biosphere, though their metabolic capabilities have remained elusive. In contrast to previously examined Chloroflexi SAGs, our genomes (several are from the order Anaerolineales) were recovered from a hydrothermally driven system and therefore provide a unique window into

  9. Importance of c-Type cytochromes for U(VI reduction by Geobacter sulfurreducens

    Directory of Open Access Journals (Sweden)

    Leang Ching

    2007-03-01

    Full Text Available Abstract Background In order to study the mechanism of U(VI reduction, the effect of deleting c-type cytochrome genes on the capacity of Geobacter sulfurreducens to reduce U(VI with acetate serving as the electron donor was investigated. Results The ability of several c-type cytochrome deficient mutants to reduce U(VI was lower than that of the wild type strain. Elimination of two confirmed outer membrane cytochromes and two putative outer membrane cytochromes significantly decreased (ca. 50–60% the ability of G. sulfurreducens to reduce U(VI. Involvement in U(VI reduction did not appear to be a general property of outer membrane cytochromes, as elimination of two other confirmed outer membrane cytochromes, OmcB and OmcC, had very little impact on U(VI reduction. Among the periplasmic cytochromes, only MacA, proposed to transfer electrons from the inner membrane to the periplasm, appeared to play a significant role in U(VI reduction. A subpopulation of both wild type and U(VI reduction-impaired cells, 24–30%, accumulated amorphous uranium in the periplasm. Comparison of uranium-accumulating cells demonstrated a similar amount of periplasmic uranium accumulation in U(VI reduction-impaired and wild type G. sulfurreducens. Assessment of the ability of the various suspensions to reduce Fe(III revealed no correlation between the impact of cytochrome deletion on U(VI reduction and reduction of Fe(III hydroxide and chelated Fe(III. Conclusion This study indicates that c-type cytochromes are involved in U(VI reduction by Geobacter sulfurreducens. The data provide new evidence for extracellular uranium reduction by G. sulfurreducens but do not rule out the possibility of periplasmic uranium reduction. Occurrence of U(VI reduction at the cell surface is supported by the significant impact of elimination of outer membrane cytochromes on U(VI reduction and the lack of correlation between periplasmic uranium accumulation and the capacity for uranium

  10. Pro-apoptotic effect of a Mycoplasma hyopneumoniae putative type I signal peptidase on PK(15) swine cells.

    Science.gov (United States)

    Paes, Jéssica A; Virginio, Veridiana G; Cancela, Martín; Leal, Fernanda M A; Borges, Thiago J; Jaeger, Natália; Bonorino, Cristina; Schrank, Irene S; Ferreira, Henrique B

    2017-03-01

    Mycoplasma hyopneumoniae is an economically significant swine pathogen that causes porcine enzootic pneumonia (PEP). Important processes for swine infection by M. hyopneumoniae depend on cell surface proteins, many of which are secreted by secretion pathways not completely elucidated so far. A putative type I signal peptidase (SPase I), a possible component of a putative Sec-dependent pathway, was annotated as a product of the sipS gene in the pathogenic M. hyopneumoniae 7448 genome. This M. hyopneumoniae putative SPase I (MhSPase I) displays only 14% and 23% of sequence identity/similarity to Escherichia coli bona fide SPase I, and, in complementation assays performed with a conditional E. coli SPase I mutant, only a partial restoration of growth was achieved with the heterologous expression of a recombinant MhSPase I (rMhSPase I). Considering the putative surface location of MhSPase I and its previously demonstrated capacity to induce a strong humoral response, we then assessed its potential to elicit a cellular and possible immunomodulatory response. In assays for immunogenicity assessment, rMhSPase I unexpectedly showed a cytotoxic effect on murine splenocytes. This cytotoxic effect was further confirmed using the swine epithelial PK(15) cell line in MTT and annexin V-flow cytometry assays, which showed that rMhSPase I induces apoptosis in a dose dependent-way. It was also demonstrated that this pro-apoptotic effect of rMhSPase I involves activation of a caspase-3 cascade. The potential relevance of the rMhSPase I pro-apoptotic effect for M. hyopneumoniae-host interactions in the context of PEP is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Purification and crystallization of a putative transcriptional regulator of the benzoate oxidation pathway in Burkholderia xenovorans LB400

    International Nuclear Information System (INIS)

    Law, Adrienne M.; Bains, Jasleen; Boulanger, Martin J.

    2009-01-01

    The X-ray diffraction and preliminary phasing of the putative transcriptional regulator Bxe-C0898 from B. xenovorans LB400 are reported. Burkholderia xenovorans LB400 harbours two paralogous copies of the recently discovered benzoate oxidation (box) pathway. While both copies are functional, the paralogues are differentially regulated and flanked by putative transcriptional regulators from distinct families. The putative LysR-type transcriptional regulator (LTTR) adjacent to the megaplasmid-encoded box enzymes, Bxe-C0898, has been produced recombinantly in Escherichia coli and purified to homogeneity. Gel-filtration studies show that Bxe-C0898 is a tetramer in solution, consistent with previously characterized LTTRs. Bxe-C0898 crystallized with four molecules in the asymmetric unit of the P4 3 2 1 2/P4 1 2 1 2 unit cell with a solvent content of 61.19%, as indicated by processing of the X-ray diffraction data. DNA-protection assays are currently under way in order to identify potential operator regions for this LTTR and to define its role in regulation of the box pathway

  12. Five putative nucleoside triphosphate diphosphohydrolase genes are expressed in Trichomonas vaginalis.

    Science.gov (United States)

    Frasson, Amanda Piccoli; Dos Santos, Odelta; Meirelles, Lúcia Collares; Macedo, Alexandre José; Tasca, Tiana

    2016-01-01

    Trichomonas vaginalis is a protozoan that parasitizes the human urogenital tract causing trichomoniasis, the most common non-viral sexually transmitted disease. The parasite has unique genomic characteristics such as a large genome size and expanded gene families. Ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) is an enzyme responsible for hydrolyzing nucleoside tri- and diphosphates and has already been biochemically characterized in T. vaginalis. Considering the important role of this enzyme in the production of extracellular adenosine for parasite uptake, we evaluated the gene expression of five putative NTPDases in T. vaginalis. We showed that all five putative TvNTPDase genes (TvNTPDase1-5) were expressed by both fresh clinical and long-term grown isolates. The amino acid alignment predicted the presence of the five crucial apyrase conserved regions, transmembrane domains, signal peptides, phosphorylation and catalytic sites. Moreover, a phylogenetic analysis showed that TvNTPDase sequences make up a clade with NTPDases intracellularly located. Biochemical NTPDase activity (ATP and ADP hydrolysis) is responsive to the serum-restrictive conditions and the gene expression of TvNTPDases was mostly increased, mainly TvNTPDase2 and TvNTPDase4, although there was not a clear pattern of expression among them. In summary, the present report demonstrates the gene expression patterns of predicted NTPDases in T. vaginalis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. [Detection of putative polysaccharide biosynthesis genes in Azospirillum brasilense strains from serogroups I and II].

    Science.gov (United States)

    Petrova, L P; Prilipov, A G; Katsy, E I

    2017-01-01

    It is known that in Azospirillum brasilense strains Sp245 and SR75 included in serogroup I, the repeat units of their O-polysaccharides consist of five residues of D-rhamnose, and in strain SR15, of four; and the heteropolymeric O-polysaccharide of A. brasilense type strain Sp7 from serogroup II contains not less than five types of repeat units. In the present work, a complex of nondegenerate primers to the genes of A. brasilense Sp245 plasmids AZOBR_p6, AZOBR_p3, and AZOBR_p2, which encode putative enzymes for the biosynthesis of core oligosaccharide and O-polysaccharide of lipopolysaccharide, capsular polysaccharides, and exopolysaccharides, was proposed. By using the designed primers, products of the expected sizes were synthesized in polymerase chain reactions on genomic DNA of A. brasilense Sp245, SR75, SR15, and Sp7 in 36, 29, 23, and 12 cases, respectively. As a result of sequencing of a number of amplicons, a high (86–99%) level of identity of the corresponding putative polysaccharide biosynthesis genes in three A. brasilense strains from serogroup I was detected. In a blotting-hybridization reaction with the biotin-labeled DNA of the A. brasilense gene AZOBR_p60122 coding for putative permease of the ABC transporter of polysaccharides, localization of the homologous gene in ~120-MDa plasmids of the bacteria A. brasilense SR15 and SR75 was revealed.

  14. An intertwined evolutionary history of methanogenic archaea and sulfate reduction.

    Directory of Open Access Journals (Sweden)

    Dwi Susanti

    Full Text Available Hydrogenotrophic methanogenesis and dissimilatory sulfate reduction, two of the oldest energy conserving respiratory systems on Earth, apparently could not have evolved in the same host, as sulfite, an intermediate of sulfate reduction, inhibits methanogenesis. However, certain methanogenic archaea metabolize sulfite employing a deazaflavin cofactor (F(420-dependent sulfite reductase (Fsr where N- and C-terminal halves (Fsr-N and Fsr-C are homologs of F(420H(2 dehydrogenase and dissimilatory sulfite reductase (Dsr, respectively. From genome analysis we found that Fsr was likely assembled from freestanding Fsr-N homologs and Dsr-like proteins (Dsr-LP, both being abundant in methanogens. Dsr-LPs fell into two groups defined by following sequence features: Group I (simplest, carrying a coupled siroheme-[Fe(4-S(4] cluster and sulfite-binding Arg/Lys residues; Group III (most complex, with group I features, a Dsr-type peripheral [Fe(4-S(4] cluster and an additional [Fe(4-S(4] cluster. Group II Dsr-LPs with group I features and a Dsr-type peripheral [Fe(4-S(4] cluster were proposed as evolutionary intermediates. Group III is the precursor of Fsr-C. The freestanding Fsr-N homologs serve as F(420H(2 dehydrogenase unit of a putative novel glutamate synthase, previously described membrane-bound electron transport system in methanogens and of assimilatory type sulfite reductases in certain haloarchaea. Among archaea, only methanogens carried Dsr-LPs. They also possessed homologs of sulfate activation and reduction enzymes. This suggested a shared evolutionary history for methanogenesis and sulfate reduction, and Dsr-LPs could have been the source of the oldest (3.47-Gyr ago biologically produced sulfide deposit.

  15. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi

    DEFF Research Database (Denmark)

    Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar

    2015-01-01

    Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim...... of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug......-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874...

  16. Complete nucleotide sequence and genome organization of Olive latent virus 3, a new putative member of the family Tymoviridae.

    Science.gov (United States)

    Alabdullah, Abdulkader; Minafra, Angelantonio; Elbeaino, Toufic; Saponari, Maria; Savino, Vito; Martelli, Giovanni P

    2010-09-01

    The complete nucleotide sequence and the genome organization were determined of a putative new member of the family Tymoviridae, tentatively named Olive latent virus 3 (OLV-3), recovered in southern Italy from a symptomless olive tree. The sequenced ssRNA genome comprises 7148 nucleotides excluding the poly(A) tail and contains four open reading frames (ORFs). ORF1 encodes a polyprotein of 221.6kDa in size, containing the conserved signatures of the methyltransferase (MTR), papain-like protease (PRO), helicase (HEL) and RNA-dependent RNA polymerase (RdRp) domains of the replication-associated proteins of positive-strand RNA viruses. ORF2 overlaps completely ORF1 and encodes a putative protein of 43.33kDa showing limited sequence similarity with the putative movement protein of Maize rayado fino virus (MRFV). ORF3 codes for a protein with predicted molecular mass of 28.46kDa, identified as the coat protein (CP), whereas ORF4 overlaps ORF3 and encodes a putative protein of 16kDa with sequence similarity to the p16 and p31 proteins of Citrus sudden death-associated virus (CSDaV) and Grapevine fleck virus (GFkV), respectively. Within the family Tymoviridae, OLV-3 genome has the closest identity level (49-52%) with members of the genus Marafivirus, from which, however, it differs because of the diverse genome organization and the presence of a single type of CP subunits. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  17. Crystallization and preliminary X-ray analysis of the receiver domain of a putative response regulator, BPSL0128, from Burkholderia pseudomallei

    International Nuclear Information System (INIS)

    Abd Aziz, Abd Ghani; Sedelnikova, Svetlana E.; Ruzheinikov, Sergey N.; Thorpe, Simon; Mohamed, Rahmah; Nathan, Sheila; Rafferty, John B.; Baker, Patrick J.; Rice, David W.

    2012-01-01

    The receiver domain of a putative response regulator from B. pseudomallei, BPSL0128, has been crystallized in a form suitable for X-ray analysis. bpsl0128, a gene encoding a putative response regulator from Burkholderia pseudomallei strain D286, has been cloned into a pETBLUE-1 vector system, overexpressed in Escherichia coli and purified. The full-length protein is degraded during purification to leave a fragment corresponding to the putative receiver domain, and crystals of this protein that diffracted to beyond 1.75 Å resolution have been grown by the hanging-drop vapour-diffusion technique using PEG 6000 as the precipitant. The crystals belonged to one of the enantiomorphic pair of space groups P3 1 21 and P3 2 21, with unit-cell parameters a = b = 65.69, c = 105.01 Å and either one or two molecules in the asymmetric unit

  18. Structure of Escherichia coli RutC, a member of the YjgF family and putative aminoacrylate peracid reductase of the rut operon

    International Nuclear Information System (INIS)

    Knapik, Aleksandra Alicja; Petkowski, Janusz Jurand; Otwinowski, Zbyszek; Cymborowski, Marcin Tadeusz; Cooper, David Robert; Chruszcz, Maksymilian; Krajewska, Wanda Małgorzata; Minor, Wladek

    2012-01-01

    The structure of the putative aminoacrylate peracid reductase RutC of the rut operon, a member of the YjgF family, is reported. RutC is the third enzyme in the Escherichia coli rut pathway of uracil degradation. RutC belongs to the highly conserved YjgF family of proteins. The structure of the RutC protein was determined and refined to 1.95 Å resolution. The crystal belonged to space group P2 1 2 1 2 and contained six molecules in the asymmetric unit. The structure was solved by SAD phasing and was refined to an R work of 19.3% (R free = 21.7%). The final model revealed that this protein has a Bacillus chorismate mutase-like fold and forms a homotrimer with a hydrophobic cavity in the center of the structure and ligand-binding clefts between two subunits. A likely function for RutC is the reduction of peroxy-aminoacrylate to aminoacrylate as a part of a detoxification process

  19. Complete Genome Sequence of an Avian Paramyxovirus Representative of Putative New Serotype 13

    OpenAIRE

    Goraichuk, Iryna; Sharma, Poonam; Stegniy, Borys; Muzyka, Denys; Pantin-Jackwood, Mary J.; Gerilovych, Anton; Solodiankin, Olexii; Bolotin, Vitaliy; Miller, Patti J.; Dimitrov, Kiril M.; Afonso, Claudio L.

    2016-01-01

    Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13.

  20. Complete Genome Sequence of an Avian Paramyxovirus Representative of Putative New Serotype 13

    Science.gov (United States)

    Goraichuk, Iryna; Sharma, Poonam; Stegniy, Borys; Muzyka, Denys; Pantin-Jackwood, Mary J.; Gerilovych, Anton; Solodiankin, Olexii; Bolotin, Vitaliy; Miller, Patti J.; Dimitrov, Kiril M.

    2016-01-01

    Here, we report the complete genome sequence of a virus of a putative new serotype of avian paramyxovirus (APMV). The virus was isolated from a white-fronted goose in Ukraine in 2011 and designated white-fronted goose/Ukraine/Askania-Nova/48-15-02/2011. The genomic characterization of the isolate suggests that it represents the novel avian paramyxovirus group APMV 13. PMID:27469958

  1. A putative ABC transporter is involved in negative regulation of biofilm formation by Listeria monocytogenes

    DEFF Research Database (Denmark)

    Zhu, Xinna; Long, Fei; Chen, Yonghui

    2008-01-01

    Listeria monocytogenes may persist for long periods in food processing environments. In some instances, this may be due to aggregation or biofilm formation. To investigate the mechanism controlling biofilm formation in the food-borne pathogen L. monocytogenes, we characterized LM-49, a mutant...... with enhanced ability of biofilm-formation generated via transposon Tn917 mutagenesis of L. monocytogenes 4b G. In this mutant, a Tn917 insertion has disrupted the coding region of the gene encoding a putative ATP binding cassette (ABC) transporter permease identical to Lmof2365_1771 (a putative ABC...... the same amount of biofilm biomass as the wild-type strain. Furthermore, transcription of the downstream lm.G_1770 was not influenced by the upstream Tn917 insertion, and the presence of Tn917 has no effect on biofilm formation. These results suggest that lm.G_1771 was solely responsible for the negative...

  2. Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri

    OpenAIRE

    Nigg, Jared C.; Nouri, Shahideh; Falk, Bryce W.

    2016-01-01

    Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri. Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera.

  3. A New Catabolic Plasmid in Xanthobacter and Starkeya spp. from a 1,2-Dichloroethane-Contaminated Site

    Science.gov (United States)

    Munro, Jacob E.; Liew, Elissa F.; Ly, Mai-Anh

    2016-01-01

    ABSTRACT 1,2-Dichloroethane (DCA) is a problematic xenobiotic groundwater pollutant. Bacteria are capable of biodegrading DCA, but the evolution of such bacteria is not well understood. In particular, the mechanisms by which bacteria acquire the key dehalogenase genes dhlA and dhlB have not been well defined. In this study, the genomic context of dhlA and dhlB was determined in three aerobic DCA-degrading bacteria (Starkeya novella strain EL1, Xanthobacter autotrophicus strain EL4, and Xanthobacter flavus strain EL8) isolated from a groundwater treatment plant (GTP). A haloalkane dehalogenase gene (dhlA) identical to the canonical dhlA gene from Xanthobacter sp. strain GJ10 was present in all three isolates, and, in each case, the dhlA gene was carried on a variant of a 37-kb circular plasmid, which was named pDCA. Sequence analysis of the repA replication initiator gene indicated that pDCA was a member of the pTAR plasmid family, related to catabolic plasmids from the Alphaproteobacteria, which enable growth on aromatics, dimethylformamide, and tartrate. Genes for plasmid replication, mobilization, and stabilization were identified, along with two insertion sequences (ISXa1 and ISPme1) which were likely to have mobilized dhlA and dhlB and played a role in the evolution of aerobic DCA-degrading bacteria. Two haloacid dehalogenase genes (dhlB1 and dhlB2) were detected in the GTP isolates; dhlB1 was most likely chromosomal and was similar to the canonical dhlB gene from strain GJ10, while dhlB2 was carried on pDCA and was not closely related to dhlB1. Heterologous expression of the DhlB2 protein confirmed that this plasmid-borne dehalogenase was capable of chloroacetate dechlorination. IMPORTANCE Earlier studies on the DCA-degrading Xanthobacter sp. strain GJ10 indicated that the key dehalogenases dhlA and dhlB were carried on a 225-kb linear plasmid and on the chromosome, respectively. The present study has found a dramatically different gene organization in more

  4. Coral bleaching under thermal stress: putative involvement of host/symbiont recognition mechanisms.

    Science.gov (United States)

    Vidal-Dupiol, Jeremie; Adjeroud, Mehdi; Roger, Emmanuel; Foure, Laurent; Duval, David; Mone, Yves; Ferrier-Pages, Christine; Tambutte, Eric; Tambutte, Sylvie; Zoccola, Didier; Allemand, Denis; Mitta, Guillaume

    2009-08-04

    Coral bleaching can be defined as the loss of symbiotic zooxanthellae and/or their photosynthetic pigments from their cnidarian host. This major disturbance of reef ecosystems is principally induced by increases in water temperature. Since the beginning of the 1980s and the onset of global climate change, this phenomenon has been occurring at increasing rates and scales, and with increasing severity. Several studies have been undertaken in the last few years to better understand the cellular and molecular mechanisms of coral bleaching but the jigsaw puzzle is far from being complete, especially concerning the early events leading to symbiosis breakdown. The aim of the present study was to find molecular actors involved early in the mechanism leading to symbiosis collapse. In our experimental procedure, one set of Pocillopora damicornis nubbins was subjected to a gradual increase of water temperature from 28 degrees C to 32 degrees C over 15 days. A second control set kept at constant temperature (28 degrees C). The differentially expressed mRNA between the stressed states (sampled just before the onset of bleaching) and the non stressed states (control) were isolated by Suppression Subtractive Hybridization. Transcription rates of the most interesting genes (considering their putative function) were quantified by Q-RT-PCR, which revealed a significant decrease in transcription of two candidates six days before bleaching. RACE-PCR experiments showed that one of them (PdC-Lectin) contained a C-Type-Lectin domain specific for mannose. Immunolocalisation demonstrated that this host gene mediates molecular interactions between the host and the symbionts suggesting a putative role in zooxanthellae acquisition and/or sequestration. The second gene corresponds to a gene putatively involved in calcification processes (Pdcyst-rich). Its down-regulation could reflect a trade-off mechanism leading to the arrest of the mineralization process under stress. Under thermal stress

  5. Two putative-aquaporin genes are differentially expressed during arbuscular mycorrhizal symbiosis in Lotus japonicus

    Directory of Open Access Journals (Sweden)

    Giovannetti Marco

    2012-10-01

    Full Text Available Abstract Background Arbuscular mycorrhizas (AM are widespread symbioses that provide great advantages to the plant, improving its nutritional status and allowing the fungus to complete its life cycle. Nevertheless, molecular mechanisms that lead to the development of AM symbiosis are not yet fully deciphered. Here, we have focused on two putative aquaporin genes, LjNIP1 and LjXIP1, which resulted to be upregulated in a transcriptomic analysis performed on mycorrhizal roots of Lotus japonicus. Results A phylogenetic analysis has shown that the two putative aquaporins belong to different functional families: NIPs and XIPs. Transcriptomic experiments have shown the independence of their expression from their nutritional status but also a close correlation with mycorrhizal and rhizobial interaction. Further transcript quantification has revealed a good correlation between the expression of one of them, LjNIP1, and LjPT4, the phosphate transporter which is considered a marker gene for mycorrhizal functionality. By using laser microdissection, we have demonstrated that one of the two genes, LjNIP1, is expressed exclusively in arbuscule-containing cells. LjNIP1, in agreement with its putative role as an aquaporin, is capable of transferring water when expressed in yeast protoplasts. Confocal analysis have demonstrated that eGFP-LjNIP1, under its endogenous promoter, accumulates in the inner membrane system of arbusculated cells. Conclusions Overall, the results have shown different functionality and expression specificity of two mycorrhiza-inducible aquaporins in L. japonicus. One of them, LjNIP1 can be considered a novel molecular marker of mycorrhizal status at different developmental stages of the arbuscule. At the same time, LjXIP1 results to be the first XIP family aquaporin to be transcriptionally regulated during symbiosis.

  6. Characterization of alternate reductant binding and electron transfer in the dopamine β-monooxygenase reaction

    International Nuclear Information System (INIS)

    Stewart, L.C.; Klinman, J.P.

    1987-01-01

    The steady-state limiting kinetic parameters V/sub max/, V/K/sub DA/, and V/K/sub O 2 /, together with deuterium isotope effects on these parameters, have been determined for the dopamine β-monooxygenase (DβM) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascrobate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of V/sub max/ and V/K/sub DA/, and the authors conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/K/sub O 2 / have been measured with [2,2- 2 H 2 ]dopamine as substrate both in the presence and in the absence of added ascorbate. This key result unambiguously rules out an entry of electrons to enzyme forms leading from the enzyme-dopamine complex to enzyme-bound product and, hence, reaction mechanisms involving a reductive activation of the putative Cu(II)-OOH prior to substrate hydroxylation

  7. Effect of Set Potential on Hexavalent Chromium Reduction and Electricity Generation from Biocathode Microbial Fuel Cells

    KAUST Repository

    Huang, Liping

    2011-06-01

    Setting a biocathode potential at ?300 mV improved the subsequent performance of an MFC for Cr(VI) reduction compared to a control (no set potential). With this set potential, the startup time was reduced to 19 days, the reduction of Cr(VI) was improved to 19.7 mg/L d, and the maximum power density was increased to 6.4 W/m3 compared to the control (26 days, 14.0 mg/L d and 4.1 W/m3). Set potentials of ?150 mV and ?300 mV also improved system performance and led to similarly higher utilization of metabolic energy gained (PMEG) than set potentials of +200 mV and ?450 mV. We observed putative pili at ?150 and ?300 mV potentials, and aggregated precipitates on bacterial surfaces in both poised and nonpoised controls. These tests show that there are optimal potentials that can be set for developing a Cr(VI) biocathode. © 2011 American Chemical Society.

  8. Effect of Set Potential on Hexavalent Chromium Reduction and Electricity Generation from Biocathode Microbial Fuel Cells

    KAUST Repository

    Huang, Liping; Chai, Xiaolei; Chen, Guohua; Logan, Bruce E

    2011-01-01

    Setting a biocathode potential at ?300 mV improved the subsequent performance of an MFC for Cr(VI) reduction compared to a control (no set potential). With this set potential, the startup time was reduced to 19 days, the reduction of Cr(VI) was improved to 19.7 mg/L d, and the maximum power density was increased to 6.4 W/m3 compared to the control (26 days, 14.0 mg/L d and 4.1 W/m3). Set potentials of ?150 mV and ?300 mV also improved system performance and led to similarly higher utilization of metabolic energy gained (PMEG) than set potentials of +200 mV and ?450 mV. We observed putative pili at ?150 and ?300 mV potentials, and aggregated precipitates on bacterial surfaces in both poised and nonpoised controls. These tests show that there are optimal potentials that can be set for developing a Cr(VI) biocathode. © 2011 American Chemical Society.

  9. Transcriptional profiling of putative human epithelial stem cells

    Directory of Open Access Journals (Sweden)

    Koçer Salih S

    2008-07-01

    Full Text Available Abstract Background Human interfollicular epidermis is sustained by the proliferation of stem cells and their progeny, transient amplifying cells. Molecular characterization of these two cell populations is essential for better understanding of self renewal, differentiation and mechanisms of skin pathogenesis. The purpose of this study was to obtain gene expression profiles of alpha 6+/MHCI+, transient amplifying cells and alpha 6+/MHCI-, putative stem cells, and to compare them with existing data bases of gene expression profiles of hair follicle stem cells. The expression of Major Histocompatibility Complex (MHC class I, previously shown to be absent in stem cells in several tissues, and alpha 6 integrin were used to isolate MHCI positive basal cells, and MHCI low/negative basal cells. Results Transcriptional profiles of the two cell populations were determined and comparisons made with published data for hair follicle stem cell gene expression profiles. We demonstrate that presumptive interfollicular stem cells, alpha 6+/MHCI- cells, are enriched in messenger RNAs encoding surface receptors, cell adhesion molecules, extracellular matrix proteins, transcripts encoding members of IFN-alpha family proteins and components of IFN signaling, but contain lower levels of transcripts encoding proteins which take part in energy metabolism, cell cycle, ribosome biosynthesis, splicing, protein translation, degradation, DNA replication, repair, and chromosome remodeling. Furthermore, our data indicate that the cell signaling pathways Notch1 and NF-κB are downregulated/inhibited in MHC negative basal cells. Conclusion This study demonstrates that alpha 6+/MHCI- cells have additional characteristics attributed to stem cells. Moreover, the transcription profile of alpha 6+/MHCI- cells shows similarities to transcription profiles of mouse hair follicle bulge cells known to be enriched for stem cells. Collectively, our data suggests that alpha 6+/MHCI- cells

  10. Characterization of Putative cis-Regulatory Elements in Genes Preferentially Expressed in Arabidopsis Male Meiocytes

    Directory of Open Access Journals (Sweden)

    Junhua Li

    2014-01-01

    Full Text Available Meiosis is essential for plant reproduction because it is the process during which homologous chromosome pairing, synapsis, and meiotic recombination occur. The meiotic transcriptome is difficult to investigate because of the size of meiocytes and the confines of anther lobes. The recent development of isolation techniques has enabled the characterization of transcriptional profiles in male meiocytes of Arabidopsis. Gene expression in male meiocytes shows unique features. The direct interaction of transcription factors (TFs with DNA regulatory sequences forms the basis for the specificity of transcriptional regulation. Here, we identified putative cis-regulatory elements (CREs associated with male meiocyte-expressed genes using in silico tools. The upstream regions (1 kb of the top 50 genes preferentially expressed in Arabidopsis meiocytes possessed conserved motifs. These motifs are putative binding sites of TFs, some of which share common functions, such as roles in cell division. In combination with cell-type-specific analysis, our findings could be a substantial aid for the identification and experimental verification of the protein-DNA interactions for the specific TFs that drive gene expression in meiocytes.

  11. VenomKB, a new knowledge base for facilitating the validation of putative venom therapies.

    Science.gov (United States)

    Romano, Joseph D; Tatonetti, Nicholas P

    2015-11-24

    Animal venoms have been used for therapeutic purposes since the dawn of recorded history. Only a small fraction, however, have been tested for pharmaceutical utility. Modern computational methods enable the systematic exploration of novel therapeutic uses for venom compounds. Unfortunately, there is currently no comprehensive resource describing the clinical effects of venoms to support this computational analysis. We present VenomKB, a new publicly accessible knowledge base and website that aims to act as a repository for emerging and putative venom therapies. Presently, it consists of three database tables: (1) Manually curated records of putative venom therapies supported by scientific literature, (2) automatically parsed MEDLINE articles describing compounds that may be venom derived, and their effects on the human body, and (3) automatically retrieved records from the new Semantic Medline resource that describe the effects of venom compounds on mammalian anatomy. Data from VenomKB may be selectively retrieved in a variety of popular data formats, are open-source, and will be continually updated as venom therapies become better understood.

  12. Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

    Science.gov (United States)

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Girginkardesler, Nogay; Celik, Pınar; Yereli, Kor; Özbilgin, Ahmet

    2014-01-01

    Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

  13. Toddlers’ Duration of Attention towards Putative Threat

    Science.gov (United States)

    Kiel, Elizabeth J.; Buss, Kristin A.

    2010-01-01

    Although individual differences in reactions to novelty in the toddler years have been consistently linked to risk for developing anxious behavior, toddlers’ attention towards a novel, putatively threatening stimulus while in the presence of other enjoyable activities has rarely been examined as a precursor to such risk. The current study examined how attention towards an angry-looking gorilla mask in a room with alternative opportunities for play in 24-month-old toddlers predicted social inhibition when children entered kindergarten. Analyses examined attention to threat above and beyond and in interaction with both proximity to the mask and fear of novelty observed in other situations. Attention to threat interacted with proximity to the mask to predict social inhibition, such that attention to threat most strongly predicted social inhibition when toddlers stayed furthest from the mask. This relation occurred above and beyond the predictive relation between fear of novelty and social inhibition. Results are discussed within the broader literature of anxiety development and attentional processes in young children. PMID:21373365

  14. Effects of drugs of abuse on putative rostromedial tegmental neurons, inhibitory afferents to midbrain dopamine cells.

    Science.gov (United States)

    Lecca, Salvatore; Melis, Miriam; Luchicchi, Antonio; Ennas, Maria Grazia; Castelli, Maria Paola; Muntoni, Anna Lisa; Pistis, Marco

    2011-02-01

    Recent findings have underlined the rostromedial tegmental nucleus (RMTg), a structure located caudally to the ventral tegmental area, as an important site involved in the mechanisms of aversion. RMTg contains γ-aminobutyric acid neurons responding to noxious stimuli, densely innervated by the lateral habenula and providing a major inhibitory projection to reward-encoding midbrain dopamine (DA) neurons. One of the key features of drug addiction is the perseverance of drug seeking in spite of negative and unpleasant consequences, likely mediated by response suppression within neural pathways mediating aversion. To investigate whether the RMTg has a function in the mechanisms of addicting drugs, we studied acute effects of morphine, cocaine, the cannabinoid agonist WIN55212-2 (WIN), and nicotine on putative RMTg neurons. We utilized single unit extracellular recordings in anesthetized rats and whole-cell patch-clamp recordings in brain slices to identify and characterize putative RMTg neurons and their responses to drugs of abuse. Morphine and WIN inhibited both firing rate in vivo and excitatory postsynaptic currents (EPSCs) evoked by stimulation of rostral afferents in vitro, whereas cocaine inhibited discharge activity without affecting EPSC amplitude. Conversely, nicotine robustly excited putative RMTg neurons and enhanced EPSCs, an effect mediated by α7-containing nicotinic acetylcholine receptors. Our results suggest that activity of RMTg neurons is profoundly influenced by drugs of abuse and, as important inhibitory afferents to midbrain DA neurons, they might take place in the complex interplay between the neural circuits mediating aversion and reward.

  15. Protein (Cyanobacteria): 652400677 [PGDBj - Ortholog DB

    Lifescience Database Archive (English)

    Full Text Available WP_026796473.1 ... 1117:3549 ... 1150:52595 1301283:73827 ... 54304:285 54307:343 ... haloacid dehalogenase Plankt...LRDVVQKFGERLGFSPTPTELESLANSIQDWQPFPDTIAALKALKQKYKLVIISNIDDNLFAQTNQHLQIEFDHIITAQQAQSYKPSAHNFQFALNKTGLSSDKLLHVAQSIFHDIATANSLGLTTVWVNRRQGQPGGGATKAAIAQPDLEVPDLKSLVDLIFEV

  16. Phylogenetic and comparative gene expression analysis of barley (Hordeum vulgare)WRKY transcription factor family reveals putatively retained functions betweenmonocots and dicots

    Energy Technology Data Exchange (ETDEWEB)

    Mangelsen, Elke; Kilian, Joachim; Berendzen, Kenneth W.; Kolukisaoglu, Uner; Harter, Klaus; Jansson, Christer; Wanke, Dierk

    2008-02-01

    WRKY proteins belong to the WRKY-GCM1 superfamily of zinc finger transcription factors that have been subject to a large plant-specific diversification. For the cereal crop barley (Hordeum vulgare), three different WRKY proteins have been characterized so far, as regulators in sucrose signaling, in pathogen defense, and in response to cold and drought, respectively. However, their phylogenetic relationship remained unresolved. In this study, we used the available sequence information to identify a minimum number of 45 barley WRKY transcription factor (HvWRKY) genes. According to their structural features the HvWRKY factors were classified into the previously defined polyphyletic WRKY subgroups 1 to 3. Furthermore, we could assign putative orthologs of the HvWRKY proteins in Arabidopsis and rice. While in most cases clades of orthologous proteins were formed within each group or subgroup, other clades were composed of paralogous proteins for the grasses and Arabidopsis only, which is indicative of specific gene radiation events. To gain insight into their putative functions, we examined expression profiles of WRKY genes from publicly available microarray data resources and found group specific expression patterns. While putative orthologs of the HvWRKY transcription factors have been inferred from phylogenetic sequence analysis, we performed a comparative expression analysis of WRKY genes in Arabidopsis and barley. Indeed, highly correlative expression profiles were found between some of the putative orthologs. HvWRKY genes have not only undergone radiation in monocot or dicot species, but exhibit evolutionary traits specific to grasses. HvWRKY proteins exhibited not only sequence similarities between orthologs with Arabidopsis, but also relatedness in their expression patterns. This correlative expression is indicative for a putative conserved function of related WRKY proteins in mono- and dicot species.

  17. Complete Genome Sequence of a Putative Densovirus of the Asian Citrus Psyllid, Diaphorina citri.

    Science.gov (United States)

    Nigg, Jared C; Nouri, Shahideh; Falk, Bryce W

    2016-07-28

    Here, we report the complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri Diaphorina citri densovirus (DcDNV) was originally identified through metagenomics, and here, we obtained the complete nucleotide sequence using PCR-based approaches. Phylogenetic analysis places DcDNV between viruses of the Ambidensovirus and Iteradensovirus genera. Copyright © 2016 Nigg et al.

  18. De Novo assembly of the Japanese flounder (Paralichthys olivaceus spleen transcriptome to identify putative genes involved in immunity.

    Directory of Open Access Journals (Sweden)

    Lin Huang

    Full Text Available Japanese flounder (Paralichthys olivaceus is an economically important marine fish in Asia and has suffered from disease outbreaks caused by various pathogens, which requires more information for immune relevant genes on genome background. However, genomic and transcriptomic data for Japanese flounder remain scarce, which limits studies on the immune system of this species. In this study, we characterized the Japanese flounder spleen transcriptome using an Illumina paired-end sequencing platform to identify putative genes involved in immunity.A cDNA library from the spleen of P. olivaceus was constructed and randomly sequenced using an Illumina technique. The removal of low quality reads generated 12,196,968 trimmed reads, which assembled into 96,627 unigenes. A total of 21,391 unigenes (22.14% were annotated in the NCBI Nr database, and only 1.1% of the BLASTx top-hits matched P. olivaceus protein sequences. Approximately 12,503 (58.45% unigenes were categorized into three Gene Ontology groups, 19,547 (91.38% were classified into 26 Cluster of Orthologous Groups, and 10,649 (49.78% were assigned to six Kyoto Encyclopedia of Genes and Genomes pathways. Furthermore, 40,928 putative simple sequence repeats and 47, 362 putative single nucleotide polymorphisms were identified. Importantly, we identified 1,563 putative immune-associated unigenes that mapped to 15 immune signaling pathways.The P. olivaceus transciptome data provides a rich source to discover and identify new genes, and the immune-relevant sequences identified here will facilitate our understanding of the mechanisms involved in the immune response. Furthermore, the plentiful potential SSRs and SNPs found in this study are important resources with respect to future development of a linkage map or marker assisted breeding programs for the flounder.

  19. Evaluation of two putative susceptibility loci for oral clefts in the Danish population

    DEFF Research Database (Denmark)

    Mitchell, L E; Murray, J C; O'Brien, S

    2001-01-01

    . The present study evaluated potential associations between CL+/-P and CP and two putative clefting susceptibility loci, MSX1 and TGFB3, using data from a nationwide case-control study conducted in Denmark from 1991 to 1994. The potential effects of interactions between these genes and two common environmental......-environment interactions involving MSX1 or TGFB3 and either first trimester exposure to maternal cigarette smoke or alcohol consumption....

  20. New record of Scedosporium dehoogii from Chile: Phylogeny and susceptibility profiles to classic and novel putative antifungal agents.

    Science.gov (United States)

    Alvarez, Eduardo; Sanhueza, Camila

    Scedosporium species are considered emerging agents causing illness in immunocompromised patients. In Chile, only Scedosporium apiospermum, Scedosporium boydii and Lomentospora prolificans haven been reported previously. The study aimed to characterize genetically Scedosporium dehoogii strains from Chilean soil samples, and assessed the antifungal susceptibility profile to classic and novel putative antifungal molecules. In 2014, several samples were obtained during a survey of soil fungi in urban areas from Chile. Morphological and phylogenetic analyses of the internal transcribed spacer region (ITS), tubulin (TUB), and calmodulin (CAL) sequences were performed. In addition, the susceptibility profiles to classic antifungal and new putative antifungal molecules were determined. Four strains of Scedosporium dehoogii were isolated from soil samples. The methodology confirmed the species (reported here as a new record for Chile). Antifungal susceptibility testing demonstrates the low activity of terpenes (α-pinene and geraniol) against this species. Voriconazole (VRC), posaconazole (PSC), and the hydroxyquinolines (clioquinol, and 5,7-dibromo-8-hydroxyquinoline) showed the best antifungal activity. Our results demonstrate that Scedosporium dehoogii is present in soil samples from Chile. This study shows also that hydroxyquinolines have potential as putative antifungal molecules. Copyright © 2016 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

  1. Hepatology may have problems with putative surrogate outcome measures

    DEFF Research Database (Denmark)

    Gluud, Christian; Brok, Jesper; Gong, Yan

    2007-01-01

    A surrogate outcome measure is a laboratory measurement, a physical sign, or another intermediate substitute that is able to predict an intervention's effect on a clinically meaningful outcome. A clinical outcome detects how a patient feels, functions, or survives. Surrogate outcome measures occur...... faster or more often, are cheaper, and/or are less invasively achieved than the clinical outcome. In practice, validation is surprisingly often overlooked, especially if a biologic plausible rationale is proposed. Surrogate outcomes must be validated before use. The first step in validation...... predicts the intervention's effect on the clinical outcome. In hepatology a number of putative surrogate outcomes are used both in clinical research and in clinical practice without having been properly validated. Sustained virological response to interferons and ribavirin in patients with chronic...

  2. Detection of putatively thermophilic anaerobic methanotrophs in diffuse hydrothermal vent fluids.

    Science.gov (United States)

    Merkel, Alexander Y; Huber, Julie A; Chernyh, Nikolay A; Bonch-Osmolovskaya, Elizaveta A; Lebedinsky, Alexander V

    2013-02-01

    The anaerobic oxidation of methane (AOM) is carried out by a globally distributed group of uncultivated Euryarchaeota, the anaerobic methanotrophic arachaea (ANME). In this work, we used G+C analysis of 16S rRNA genes to identify a putatively thermophilic ANME group and applied newly designed primers to study its distribution in low-temperature diffuse vent fluids from deep-sea hydrothermal vents. We found that the G+C content of the 16S rRNA genes (P(GC)) is significantly higher in the ANME-1GBa group than in other ANME groups. Based on the positive correlation between the P(GC) and optimal growth temperatures (T(opt)) of archaea, we hypothesize that the ANME-1GBa group is adapted to thrive at high temperatures. We designed specific 16S rRNA gene-targeted primers for the ANME-1 cluster to detect all phylogenetic groups within this cluster, including the deeply branching ANME-1GBa group. The primers were successfully tested both in silico and in experiments with sediment samples where ANME-1 phylotypes had previously been detected. The primers were further used to screen for the ANME-1 microorganisms in diffuse vent fluid samples from deep-sea hydrothermal vents in the Pacific Ocean, and sequences belonging to the ANME-1 cluster were detected in four individual vents. Phylotypes belonging to the ANME-1GBa group dominated in clone libraries from three of these vents. Our findings provide evidence of existence of a putatively extremely thermophilic group of methanotrophic archaea that occur in geographically and geologically distinct marine hydrothermal habitats.

  3. Identification and phylogenetic analysis of Tityus pachyurus and Tityus obscurus novel putative Na+-channel scorpion toxins.

    Directory of Open Access Journals (Sweden)

    Jimmy A Guerrero-Vargas

    Full Text Available Colombia and Brazil are affected by severe cases of scorpionism. In Colombia the most dangerous accidents are caused by Tityus pachyurus that is widely distributed around this country. In the Brazilian Amazonian region scorpion stings are a common event caused by Tityus obscurus. The main objective of this work was to perform the molecular cloning of the putative Na(+-channel scorpion toxins (NaScTxs from T. pachyurus and T. obscurus venom glands and to analyze their phylogenetic relationship with other known NaScTxs from Tityus species.cDNA libraries from venom glands of these two species were constructed and five nucleotide sequences from T. pachyurus were identified as putative modulators of Na(+-channels, and were named Tpa4, Tpa5, Tpa6, Tpa7 and Tpa8; the latter being the first anti-insect excitatory β-class NaScTx in Tityus scorpion venom to be described. Fifteen sequences from T. obscurus were identified as putative NaScTxs, among which three had been previously described, and the others were named To4 to To15. The peptides Tpa4, Tpa5, Tpa6, To6, To7, To9, To10 and To14 are closely related to the α-class NaScTxs, whereas Tpa7, Tpa8, To4, To8, To12 and To15 sequences are more related to the β-class NaScTxs. To5 is possibly an arthropod specific toxin. To11 and To13 share sequence similarities with both α and β NaScTxs. By means of phylogenetic analysis using the Maximum Parsimony method and the known NaScTxs from Tityus species, these toxins were clustered into 14 distinct groups.This communication describes new putative NaScTxs from T. pachyurus and T. obscurus and their phylogenetic analysis. The results indicate clear geographic separation between scorpions of Tityus genus inhabiting the Amazonian and Mountain Andes regions and those distributed over the Southern of the Amazonian rainforest. Based on the consensus sequences for the different clusters, a new nomenclature for the NaScTxs is proposed.

  4. A synthetic peptide with the putative iron binding motif of amyloid precursor protein (APP does not catalytically oxidize iron.

    Directory of Open Access Journals (Sweden)

    Kourosh Honarmand Ebrahimi

    Full Text Available The β-amyloid precursor protein (APP, which is a key player in Alzheimer's disease, was recently reported to possess an Fe(II binding site within its E2 domain which exhibits ferroxidase activity [Duce et al. 2010, Cell 142: 857]. The putative ligands of this site were compared to those in the ferroxidase site of ferritin. The activity was indirectly measured using transferrin, which scavenges the Fe(III product of the reaction. A 22-residue synthetic peptide, named FD1, with the putative ferroxidase site of APP, and the E2 domain of APP were each reported to exhibit 40% of the ferroxidase activity of APP and of ceruloplasmin. It was also claimed that the ferroxidase activity of APP is inhibited by Zn(II just as in ferritin. We measured the ferroxidase activity indirectly (i by the incorporation of the Fe(III product of the ferroxidase reaction into transferrin and directly (ii by monitoring consumption of the substrate molecular oxygen. The results with the FD1 peptide were compared to the established ferroxidase activities of human H-chain ferritin and of ceruloplasmin. For FD1 we observed no activity above the background of non-enzymatic Fe(II oxidation by molecular oxygen. Zn(II binds to transferrin and diminishes its Fe(III incorporation capacity and rate but it does not specifically bind to a putative ferroxidase site of FD1. Based on these results, and on comparison of the putative ligands of the ferroxidase site of APP with those of ferritin, we conclude that the previously reported results for ferroxidase activity of FD1 and - by implication - of APP should be re-evaluated.

  5. Exploring Universal Partnerships and Putative Marriages as Tools for Awarding Partnership Property in Contemporary Family Law

    Directory of Open Access Journals (Sweden)

    Elsje Bonthuys

    2016-12-01

    Full Text Available Following upon the Supreme Court of Appeal's judgment in Butters v Mncora 2012 4 SA 1 (SCA, which broadened the criteria and consequences of universal partnerships in cohabitation relationships, this article investigates the potential of universal partnerships and putative marriages to allocate rights to share in partnership property in other intimate relationships. It traverses several instances in which marriages are not recognised - bigamous marriages, Muslim and Hindu religious marriages and invalid customary marriages – examining whether the wives in these marriages could use universal partnerships and putative marriages to claim a share in property. It then considers the use of universal partnerships to obtain a share of property in civil marriages out of community of property. It concludes by pointing out several issues which are in need of clarification and where the common law should be developed to give effect to fundamental constitutional rights.

  6. Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis

    International Nuclear Information System (INIS)

    Stsiapanava, Alena; Koudelakova, Tana; Lapkouski, Mikalai; Pavlova, Martina; Damborsky, Jiri; Kuta Smatanova, Ivana

    2008-01-01

    Three mutants of the haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 were crystallized and diffracted to ultrahigh resolution. The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2 1 2 1 2 1 , while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 Å, respectively

  7. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane

    International Nuclear Information System (INIS)

    Lahoda, Maryna; Chaloupkova, Radka; Stsiapanava, Alena; Damborsky, Jiri; Kuta Smatanova, Ivana

    2011-01-01

    A mutant of the haloalkane dehalogenase DhaA (DhaA31) from R. rhodochrous NCIMB 13064 and its complex with 1,2,3-trichloropropane were crystallized and the crystals diffracted to high resolution. Haloalkane dehalogenases hydrolyze carbon–halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure–function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP

  8. Development of an enzymatic fiber-optic biosensor for detection of halogenated hydrocarbons

    Energy Technology Data Exchange (ETDEWEB)

    Bidmanova, Sarka; Chaloupkova, Radka; Damborsky, Jiri; Prokop, Zbynek [Masaryk University, Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment, Faculty of Science, Brno (Czech Republic)

    2010-11-15

    An enzyme-based biosensor was developed by co-immobilization of purified enzyme haloalkane dehalogenase (EC 3.8.1.5) and a fluorescence pH indicator on the tip of an optical fiber. Haloalkane dehalogenase catalyzes hydrolytic dehalogenation of halogenated aliphatic hydrocarbons, which is accompanied by a pH change influencing the fluorescence of the indicator. The pH sensitivity of several fluorescent dyes was evaluated. The selected indicator 5(6)-carboxyfluorescein was conjugated with bovine serum albumin and its reaction was tested under different immobilization conditions. The biosensor was prepared by cross-linking of the conjugate in tandem with haloalkane dehalogenase using glutaraldehyde vapor. The biosensor, stored for 24 h in 50 mM phosphate buffer (pH 7.5) prior to measurement, was used after 15 min of equilibration, the halogenated compound was added, and the response was monitored for 30 min. Calibration of the biosensor with 1,2-dibromoethane and 3-chloro-2-(chloromethyl)-1-propene showed an excellent linear dependence, with detection limits of 0.133 and 0.014 mM, respectively. This biosensor provides a new tool for continuous in situ monitoring of halogenated environmental pollutants. (orig.)

  9. TLR4, NOD1 and NOD2 mediate immune recognition of putative newly identified periodontal pathogens.

    Science.gov (United States)

    Marchesan, Julie; Jiao, Yizu; Schaff, Riley A; Hao, Jie; Morelli, Thiago; Kinney, Janet S; Gerow, Elizabeth; Sheridan, Rachel; Rodrigues, Vinicius; Paster, Bruce J; Inohara, Naohiro; Giannobile, William V

    2016-06-01

    Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. Although the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, six being classical pathogens and four putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone-marrow-derived macrophages (BMDM) from wild-type (WT) and Toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. Campylobacter concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2 stimulatory activity. These studies allowed us to provide important evidence on newly identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. TLR4, NOD1 and NOD2 Mediate Immune Recognition of Putative Newly-Identified Periodontal Pathogens

    Science.gov (United States)

    Schaff, Riley A.; Hao, Jie; Morelli, Thiago; Kinney, Janet S.; Gerow, Elizabeth; Sheridan, Rachel; Rodrigues, Vinicius; Paster, Bruce J.; Inohara, Naohiro; Giannobile, William V.

    2015-01-01

    SUMMARY Periodontitis is a polymicrobial inflammatory disease that results from the interaction between the oral microbiota and the host immunity. While the innate immune response is important for disease initiation and progression, the innate immune receptors that recognize both classical and putative periodontal pathogens that elicit an immune response have not been elucidated. By using the Human Oral Microbe Identification Microarray (HOMIM), we identified multiple predominant oral bacterial species in human plaque biofilm that strongly associate with severe periodontitis. Ten of the identified species were evaluated in greater depth, 6 being classical pathogens and 4 putative novel pathogens. Using human peripheral blood monocytes (HPBM) and murine bone marrow–derived macrophages (BMDM) from wild-type (WT) and toll-like receptor (TLR)-specific and MyD88 knockouts (KOs), we demonstrated that heat-killed Campylobacter concisus, Campylobacter rectus, Selenomonas infelix, Porphyromonas endodontalis, Porphyromonas gingivalis, and Tannerella forsythia mediate high immunostimulatory activity. C. concisus, C. rectus, and S. infelix exhibited robust TLR4 stimulatory activity. Studies using mesothelial cells from WT and NOD1-specific KOs and NOD2-expressing human embryonic kidney (HEK) cells demonstrated that Eubacterium saphenum, Eubacterium nodatum and Filifactor alocis exhibit robust NOD1 stimulatory activity, and that Porphyromonas endodontalis and Parvimonas micra have the highest NOD2-stimulatory activity. These studies allowed us to provide important evidence on newly-identified putative pathogens in periodontal disease pathogenesis showing that these bacteria exhibit different immunostimulatory activity via TLR4, NOD1, and NOD2 (Clinicaltrials.gov NCT01154855). PMID:26177212

  11. Hyla chrysoscelis (Cope’s gray treefrog) x Hyla cinerea (green treefrog): putative natural hybrid

    Science.gov (United States)

    Glorioso, Brad M.; Waddle, J. Hardin; Jenkins, Jill A.; Olivier, Heather M.; Layton, Rebekah R.

    2015-01-01

    Naturally–occurring hybrid treefrogs have been occasionally found in the eastern United States. However, these hybrids are almost always between members of the same species group. On 10 Jun 2014, at 2145 h, we located an individual making an unusual advertisement call along Bayou Manual Road in Sherburne Wildlife Management Area in the Atchafalaya Basin of south-central Louisiana, USA, and brought it back to the laboratory for further study. Physically, the treefrog appeared intermediate between a Green Treefrog and a Cope’s Gray Treefrog, which are members of different species groups. Call analysis also showed the individual to be intermediate between the two putative parental species. Flow cytometry was used to estimate the total genome size from nuclei of whole blood cells, and also determined the individual to be intermediate of the putative parental species. Despite vocalizing for mates, the hybrid did not appear to have viable spermatozoa, and was likely the result of an anomalous mis-mating event between a male Cope’s Gray Treefrog and a female Green Treefrog. To our knowledge, natural hybrids between a Cope’s Gray Treefrog and a Green Treefrog have not been previously reported.

  12. Coral bleaching under thermal stress: putative involvement of host/symbiont recognition mechanisms

    Directory of Open Access Journals (Sweden)

    Tambutte Sylvie

    2009-08-01

    Full Text Available Abstract Background Coral bleaching can be defined as the loss of symbiotic zooxanthellae and/or their photosynthetic pigments from their cnidarian host. This major disturbance of reef ecosystems is principally induced by increases in water temperature. Since the beginning of the 1980s and the onset of global climate change, this phenomenon has been occurring at increasing rates and scales, and with increasing severity. Several studies have been undertaken in the last few years to better understand the cellular and molecular mechanisms of coral bleaching but the jigsaw puzzle is far from being complete, especially concerning the early events leading to symbiosis breakdown. The aim of the present study was to find molecular actors involved early in the mechanism leading to symbiosis collapse. Results In our experimental procedure, one set of Pocillopora damicornis nubbins was subjected to a gradual increase of water temperature from 28°C to 32°C over 15 days. A second control set kept at constant temperature (28°C. The differentially expressed mRNA between the stressed states (sampled just before the onset of bleaching and the non stressed states (control were isolated by Suppression Subtractive Hybridization. Transcription rates of the most interesting genes (considering their putative function were quantified by Q-RT-PCR, which revealed a significant decrease in transcription of two candidates six days before bleaching. RACE-PCR experiments showed that one of them (PdC-Lectin contained a C-Type-Lectin domain specific for mannose. Immunolocalisation demonstrated that this host gene mediates molecular interactions between the host and the symbionts suggesting a putative role in zooxanthellae acquisition and/or sequestration. The second gene corresponds to a gene putatively involved in calcification processes (Pdcyst-rich. Its down-regulation could reflect a trade-off mechanism leading to the arrest of the mineralization process under stress

  13. Asymmetric total synthesis of a putative sex pheromone component from the parasitoid wasp Trichogramma turkestanica

    NARCIS (Netherlands)

    Geerdink, Danny; Buter, Jeffrey; van Beek, Teris A.; Minnaard, Adriaan J.

    2014-01-01

    Virgin females of the parasitoid wasp Trichogramma turkestanica produce minute amounts of a sex pheromone, the identity of which has not been fully established. The enantioselective synthesis of a putative component of this pheromone, (6S,8S,10S)-4,6,8,10-tetramethyltrideca-2E,4E-dien-1-ol (2), is

  14. Duplications and losses in gene families of rust pathogens highlight putative effectors.

    Science.gov (United States)

    Pendleton, Amanda L; Smith, Katherine E; Feau, Nicolas; Martin, Francis M; Grigoriev, Igor V; Hamelin, Richard; Nelson, C Dana; Burleigh, J Gordon; Davis, John M

    2014-01-01

    Rust fungi are a group of fungal pathogens that cause some of the world's most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host's cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of 16 diverse fungal species, which include 15 basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: (i) arose or expanded in rust pathogens relative to other fungi, or (ii) contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity.

  15. Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

    Science.gov (United States)

    Ciok, Anna; Adamczuk, Marcin; Bartosik, Dariusz; Dziewit, Lukasz

    2016-11-28

    Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas .

  16. Ticks and tick-borne pathogens and putative symbionts of black bears (Ursus americanus floridanus) from Georgia and Florida.

    Science.gov (United States)

    Yabsley, Michael J; Nims, Todd N; Savage, Mason Y; Durden, Lance A

    2009-10-01

    Ticks were collected from 38 black bears (Ursus americanus floridanus) from northwestern Florida (n = 18) from 2003 to 2005 and southern Georgia (n = 20) in 2006. Five species (Amblyomma americanum, A. maculatum, Dermacentor variabilis, Ixodes scapularis, and I. affinis) were collected from Florida bears, and 4 species (A. americanum, A. maculatum, D. variabilis, I. scapularis) were collected from bears in Georgia. Ixodes scapularis was the most frequently collected tick, followed by D. variabilis, A. americanum, A. maculatum, and I. affinis. The collection of I. affinis from a Florida bear represents a new host record. A subset of ticks was screened for pathogens and putative symbionts by polymerase chain reaction (PCR). The zoonotic tick-borne pathogens Ehrlichia chaffeensis and Rickettsia parkeri were detected in 1 of 23 (4.3%) A. americanum and 1 of 12 (8.3%) A. maculatum, respectively. The putative zoonotic pathogen "Rickettsia amblyommii" was detected in 4 (17.4%) A. americanum and 1 (8.3%) A. maculatum. Other putative symbiotic rickettsiae detected included R. bellii and R. montanensis in D. variabilis, a Rickettsia cooleyi-like sp. and Rickettsia sp. Is-1 in I. scapularis, and Rickettsia TR39-like sp. in I. scapularis and A. americanum. All ticks were PCR-negative for Anaplasma phagocytophilum, Panola Mountain Ehrlichia sp., E. ewingii, Francisella tularensis, and Borrelia spp.

  17. Search strings for the study of putative occupational determinants of disease

    Science.gov (United States)

    Mattioli, Stefano; Zanardi, Francesca; Baldasseroni, Alberto; Schaafsma, Frederieke; Cooke, Robin MT; Mancini, Gianpiero; Fierro, Mauro; Santangelo, Chiara; Farioli, Andrea; Fucksia, Serenella; Curti, Stefania; Verbeek, Jos

    2010-01-01

    Objective To identify efficient PubMed search strategies to retrieve articles regarding putative occupational determinants of conditions not generally considered to be work related. Methods Based on MeSH definitions and expert knowledge, we selected as candidate search terms the four MeSH terms describing ‘occupational disease’, ‘occupational exposure’, ‘occupational health’ and ‘occupational medicine’ (DEHM) alongside 22 other promising terms. We first explored overlaps between the candidate terms in PubMed. Using random samples of abstracts retrieved by each term, we estimated the proportions of articles containing potentially pertinent information regarding occupational aetiology in order to formulate two search strategies (one more ‘specific’, one more ‘sensitive’). We applied these strategies to retrieve information on the possible occupational aetiology of meningioma, pancreatitis and atrial fibrillation. Results Only 20.3% of abstracts were retrieved by more than one DEHM term. The more ‘specific’ search string was based on the combination of terms that yielded the highest proportion (40%) of potentially pertinent abstracts. The more ‘sensitive’ string was based on the use of broader search fields and additional coverage provided by other search terms under study. Using the specific string, the numbers of abstracts needed to read to find one potentially pertinent article were 1.2 for meningioma, 1.9 for pancreatitis and 1.8 for atrial fibrillation. Using the sensitive strategy, the numbers needed to read were 4.4 for meningioma, 8.9 for pancreatitis and 10.5 for atrial fibrillation. Conclusions The proposed strings could help health care professionals explore putative occupational aetiology for diseases that are not generally thought to be work related. PMID:19819858

  18. Basal ganglia calcification as a putative cause for cognitive decline.

    Science.gov (United States)

    de Oliveira, João Ricardo Mendes; de Oliveira, Matheus Fernandes

    2013-01-01

    Basal ganglia calcifications (BGC) may be present in various medical conditions, such as infections, metabolic, psychiatric and neurological diseases, associated with different etiologies and clinical outcomes, including parkinsonism, psychosis, mood swings and dementia. A literature review was performed highlighting the main neuropsychological findings of BGC, with particular attention to clinical reports of cognitive decline. Neuroimaging studies combined with neuropsychological analysis show that some patients have shown progressive disturbances of selective attention, declarative memory and verbal perseveration. Therefore, the calcification process might represent a putative cause for dementia syndromes, suggesting a probable link among calcinosis, the aging process and eventually with neuronal death. The increasing number of reports available will foster a necessary discussion about cerebral calcinosis and its role in determining symptomatology in dementia patients.

  19. Proteomic data set of the organohalide-respiring Epsilonproteobacterium Sulfurospirillum multivorans adapted to tetrachloroethene and other energy substrates

    Directory of Open Access Journals (Sweden)

    Tobias Goris

    2016-09-01

    Full Text Available Sulfurospirillum multivorans is a free-living, physiologically versatile Epsilonproteobacterium able to couple the reductive dehalogenation of chlorinated and brominated ethenes to growth (organohalide respiration. We present proteomic data of S. multivorans grown with different electron donors (formate or pyruvate and electron acceptors (fumarate, nitrate, or tetrachloroethene [PCE]. To obtain information on the cellular localization of proteins, membrane extracts and soluble fractions were separated before data collection from both fractions. The proteome analysis of S. multivorans was performed by mass spectrometry (nanoLC-MS/MS. Raw data have been deposited at ProteomeXchange, “ProteomeXchange provides globally coordinated proteomics data submission and dissemination” [1], via the PRIDE partner repository with the dataset identifier PRIDE: PXD004011. The data might support further research in organohalide respiration and in the general metabolism of free-living Epsilonproteobacteria. The dataset is associated with a previously published study “Proteomics of the organohalide-respiring Epsilonproteobacterium S. multivorans adapted to tetrachloroethene and other energy substrates” [2]. Keywords: Anaerobic respiration, Epsilonproteobacteria, Nitrate respiration, Reductive dechlorination, Reductive dehalogenase

  20. Use of dimensionality reduction for structural mapping of hip joint osteoarthritis data

    International Nuclear Information System (INIS)

    Theoharatos, C; Fotopoulos, S; Boniatis, I; Panayiotakis, G; Panagiotopoulos, E

    2009-01-01

    A visualization-based, computer-oriented, classification scheme is proposed for assessing the severity of hip osteoarthritis (OA) using dimensionality reduction techniques. The introduced methodology tries to cope with the confined ability of physicians to structurally organize the entire available set of medical data into semantically similar categories and provide the capability to make visual observations among the ensemble of data using low-dimensional biplots. In this work, 18 pelvic radiographs of patients with verified unilateral hip OA are evaluated by experienced physicians and assessed into Normal, Mild and Severe following the Kellgren and Lawrence scale. Two regions of interest corresponding to radiographic hip joint spaces are determined and representative features are extracted using a typical texture analysis technique. The structural organization of all hip OA data is accomplished using distance and topology preservation-based dimensionality reduction techniques. The resulting map is a low-dimensional biplot that reflects the intrinsic organization of the ensemble of available data and which can be directly accessed by the physician. The conceivable visualization scheme can potentially reveal critical data similarities and help the operator to visually estimate their initial diagnosis. In addition, it can be used to detect putative clustering tendencies, examine the presence of data similarities and indicate the existence of possible false alarms in the initial perceptual evaluation

  1. Update of green tea interactions with cardiovascular drugs and putative mechanisms

    Directory of Open Access Journals (Sweden)

    José Pablo Werba

    2018-04-01

    Full Text Available Many patients treated with cardiovascular (CV drugs drink green tea (GT, either as a cultural tradition or persuaded of its putative beneficial effects for health. Yet, GT may affect the pharmacokinetics and pharmacodynamics of CV compounds. Novel GT-CV drug interactions were reported for rosuvastatin, sildenafil and tacrolimus. Putative mechanisms involve inhibitory effects of GT catechins at the intestinal level on influx transporters OATP1A2 or OATP2B1 for rosuvastatin, on CYP3A for sildenafil and on both CYP3A and the efflux transporter p-glycoprotein for tacrolimus. These interactions, which add to those previously described with simvastatin, nadolol and warfarin, might lead, in some cases, to reduced drug efficacy or risk of drug toxicity. Oddly, available data on GT interaction with CV compounds with a narrow therapeutic index, such as warfarin and tacrolimus, derive from single case reports. Conversely, GT interactions with simvastatin, rosuvastatin, nadolol and sildenafil were documented through pharmacokinetic studies. In these, the effect of GT or GT derivatives on drug exposure was mild to moderate, but a high inter-individual variability was observed. Further investigations, including studies on the effect of the dose and the time of GT intake are necessary to understand more in depth the clinical relevance of GT-CV drug interactions. Keywords: Cardiovascular drugs, Green tea, Herb–drug interactions

  2. Differential transcript abundance and genotypic variation of four putative allergen-encoding gene families in melting peach

    NARCIS (Netherlands)

    Yang, Z.; Ma, Y.; Chen, L.; Xie, R.; Zhang, X.; Zhang, B.; Lu, M.; Wu, S.; Gilissen, L.J.W.J.; Ree, van R.; Gao, Z.

    2011-01-01

    We analysed the temporal and spatial transcript expression of the panel of 18 putative isoallergens from four gene families (Pru p 1–4) in the peach fruit, anther and leaf of two melting cultivars, to gain insight into their expression profiles and to identify the key family members. Genotypic

  3. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

    2017-08-02

    In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  4. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Energy Technology Data Exchange (ETDEWEB)

    Forlani, Giuseppe; Nocek, Boguslaw; Chakravarthy, Srinivas; Joachimiak, Andrzej

    2017-08-02

    In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of delta(1)-pyrroline-5-carboxylate (P5C) catalyzed by P5C reductase (EC 1.5.1.2). In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER) that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  5. Functional Characterization of Four Putative δ1-Pyrroline-5-Carboxylate Reductases from Bacillus subtilis

    Directory of Open Access Journals (Sweden)

    Giuseppe Forlani

    2017-08-01

    Full Text Available In most living organisms, the amino acid proline is synthesized starting from both glutamate and ornithine. In prokaryotes, in the absence of an ornithine cyclodeaminase that has been identified to date only in a small number of soil and plant bacteria, these pathways share the last step, the reduction of δ1-pyrroline-5-carboxylate (P5C catalyzed by P5C reductase (EC 1.5.1.2. In several species, multiple forms of P5C reductase have been reported, possibly reflecting the dual function of proline. Aside from its common role as a building block of proteins, proline is indeed also involved in the cellular response to osmotic and oxidative stress conditions. Genome analysis of Bacillus subtilis identifies the presence of four genes (ProH, ProI, ProG, and ComER that, based on bioinformatic and phylogenic studies, were defined as respectively coding a putative P5C reductase. Here we describe the cloning, heterologous expression, functional analysis and small-angle X-ray scattering studies of the four affinity-purified proteins. Results showed that two of them, namely ProI and ComER, lost their catalytic efficiency or underwent subfunctionalization. In the case of ComER, this could be likely explained by the loss of the ability to form a dimer, which has been previously shown to be an essential structural feature of the catalytically active P5C reductase. The properties of the two active enzymes are consistent with a constitutive role for ProG, and suggest that ProH expression may be beneficial to satisfy an increased need for proline.

  6. Markerless deletion of putative alanine dehydrogenase genes in Bacillus licheniformis using a codBA-based counterselection technique.

    Science.gov (United States)

    Kostner, David; Rachinger, Michael; Liebl, Wolfgang; Ehrenreich, Armin

    2017-11-01

    Bacillus licheniformis strains are used for the large-scale production of industrial exoenzymes from proteinaceous substrates, but details of the amino acid metabolism involved are largely unknown. In this study, two chromosomal genes putatively involved in amino acid metabolism of B. licheniformis were deleted to clarify their role. For this, a convenient counterselection system for markerless in-frame deletions was developed for B. licheniformis. A deletion plasmid containing up- and downstream DNA segments of the chromosomal deletion target was conjugated to B. licheniformis and integrated into the genome by homologous recombination. Thereafter, the counterselection was done by using a codBA cassette. The presence of cytosine deaminase and cytosine permease exerted a conditionally lethal phenotype on B. licheniformis cells in the presence of the cytosine analogue 5-fluorocytosine. Thereby clones were selected that lost the integrated vector sequence and the anticipated deletion target after a second recombination step. This method allows the construction of markerless mutants in Bacillus strains in iterative cycles. B. licheniformis MW3 derivatives lacking either one of the ORFs BL03009 or BL00190, encoding a putative alanine dehydrogenase and a similar putative enzyme, respectively, retained the ability to grow in minimal medium supplemented with alanine as the carbon source. In the double deletion mutant MW3 ΔBL03009 ΔBL00190, however, growth on alanine was completely abolished. These data indicate that the two encoded enzymes are paralogues fulfilling mutually replaceable functions in alanine utilization, and suggest that in B. licheniformis MW3 alanine utilization is initiated by direct oxidative transamination to pyruvate and ammonium.

  7. Over-expression of a putative oxidoreductase (UcpA) for increasing furfural or 5-hydroxymethylfurfural tolerance

    Science.gov (United States)

    Wang, Xuan; Miller, Elliot N.; Yomano, Lorraine P.; Shanmugam, Keelnatham T.; Ingram, Lonnie O'Neal

    2016-05-24

    The subject invention pertains to overexpression of a putative oxidoreductase (ucpA) for increasing furfural tolerance in genetically modified microorganisms. Genetically modified microorganisms capable of overexpressing UcpA are also provided. Increased expression of ucpA was shown to increase furfural tolerance by 50%, and to permit the fermentation of sugars to products in the presence of 15 mM furfural.

  8. The Fusarium oxysporum gnt2, encoding a putative N-acetylglucosamine transferase, is involved in cell wall architecture and virulence.

    Directory of Open Access Journals (Sweden)

    Loida López-Fernández

    Full Text Available With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity.

  9. The Fusarium oxysporum gnt2, Encoding a Putative N-Acetylglucosamine Transferase, Is Involved in Cell Wall Architecture and Virulence

    Science.gov (United States)

    López-Fernández, Loida; Ruiz-Roldán, Carmen; Pareja-Jaime, Yolanda; Prieto, Alicia; Khraiwesh, Husam; Roncero, M. Isabel G.

    2013-01-01

    With the aim to decipher the molecular dialogue and cross talk between Fusarium oxysporum f.sp. lycopersci and its host during infection and to understand the molecular bases that govern fungal pathogenicity, we analysed genes presumably encoding N-acetylglucosaminyl transferases, involved in glycosylation of glycoproteins, glycolipids, proteoglycans or small molecule acceptors in other microorganisms. In silico analysis revealed the existence of seven putative N-glycosyl transferase encoding genes (named gnt) in F. oxysporum f.sp. lycopersici genome. gnt2 deletion mutants showed a dramatic reduction in virulence on both plant and animal hosts. Δgnt2 mutants had αalterations in cell wall properties related to terminal αor β-linked N-acetyl glucosamine. Mutant conidia and germlings also showed differences in structure and physicochemical surface properties. Conidial and hyphal aggregation differed between the mutant and wild type strains, in a pH independent manner. Transmission electron micrographs of germlings showed strong cell-to-cell adherence and the presence of an extracellular chemical matrix. Δgnt2 cell walls presented a significant reduction in N-linked oligosaccharides, suggesting the involvement of Gnt2 in N-glycosylation of cell wall proteins. Gnt2 was localized in Golgi-like sub-cellular compartments as determined by fluorescence microscopy of GFP::Gnt2 fusion protein after treatment with the antibiotic brefeldin A or by staining with fluorescent sphingolipid BODIPY-TR ceramide. Furthermore, density gradient ultracentrifugation allowed co-localization of GFP::Gnt2 fusion protein and Vps10p in subcellular fractions enriched in Golgi specific enzymatic activities. Our results suggest that N-acetylglucosaminyl transferases are key components for cell wall structure and influence interactions of F. oxysporum with both plant and animal hosts during pathogenicity. PMID:24416097

  10. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    Science.gov (United States)

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

  11. Transcriptome of Aphanomyces euteiches: new oomycete putative pathogenicity factors and metabolic pathways.

    Directory of Open Access Journals (Sweden)

    Elodie Gaulin

    Full Text Available Aphanomyces euteiches is an oomycete pathogen that causes seedling blight and root rot of legumes, such as alfalfa and pea. The genus Aphanomyces is phylogenically distinct from well-studied oomycetes such as Phytophthora sp., and contains species pathogenic on plants and aquatic animals. To provide the first foray into gene diversity of A. euteiches, two cDNA libraries were constructed using mRNA extracted from mycelium grown in an artificial liquid medium or in contact to plant roots. A unigene set of 7,977 sequences was obtained from 18,864 high-quality expressed sequenced tags (ESTs and characterized for potential functions. Comparisons with oomycete proteomes revealed major differences between the gene content of A. euteiches and those of Phytophthora species, leading to the identification of biosynthetic pathways absent in Phytophthora, of new putative pathogenicity genes and of expansion of gene families encoding extracellular proteins, notably different classes of proteases. Among the genes specific of A. euteiches are members of a new family of extracellular proteins putatively involved in adhesion, containing up to four protein domains similar to fungal cellulose binding domains. Comparison of A. euteiches sequences with proteomes of fully sequenced eukaryotic pathogens, including fungi, apicomplexa and trypanosomatids, allowed the identification of A. euteiches genes with close orthologs in these microorganisms but absent in other oomycetes sequenced so far, notably transporters and non-ribosomal peptide synthetases, and suggests the presence of a defense mechanism against oxidative stress which was initially characterized in the pathogenic trypanosomatids.

  12. Duplications and losses in gene families of rust pathogens highlight putative effectors

    Directory of Open Access Journals (Sweden)

    Amanda L. Pendleton

    2014-06-01

    Full Text Available Rust fungi are a group of fungal pathogens that cause some of the world’s most destructive diseases of trees and crops. A shared characteristic among rust fungi is obligate biotrophy, the inability to complete a lifecycle without a host. This dependence on a host species likely affects patterns of gene expansion, contraction, and innovation within rust pathogen genomes. The establishment of disease by biotrophic pathogens is reliant upon effector proteins that are encoded in the fungal genome and secreted from the pathogen into the host’s cell apoplast or within the cells. This study uses a comparative genomic approach to elucidate putative effectors and determine their evolutionary histories. We used OrthoMCL to identify nearly 20,000 gene families in proteomes of sixteen diverse fungal species, which include fifteen basidiomycetes and one ascomycete. We inferred patterns of duplication and loss for each gene family and identified families with distinctive patterns of expansion/contraction associated with the evolution of rust fungal genomes. To recognize potential contributors for the unique features of rust pathogens, we identified families harboring secreted proteins that: i arose or expanded in rust pathogens relative to other fungi, or ii contracted or were lost in rust fungal genomes. While the origin of rust fungi appears to be associated with considerable gene loss, there are many gene duplications associated with each sampled rust fungal genome. We also highlight two putative effector gene families that have expanded in Cqf that we hypothesize have roles in pathogenicity.

  13. Apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ.

    Directory of Open Access Journals (Sweden)

    Jiaojiao Chu

    Full Text Available Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.

  14. The Bacillus anthracis chromosome contains four conserved, excision-proficient, putative prophages

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    Sozhamannan Shanmuga

    2006-04-01

    Full Text Available Abstract Background Bacillus anthracis is considered to be a recently emerged clone within the Bacillus cereus sensu lato group. The B. anthracis genome sequence contains four putative lambdoid prophages. We undertook this study in order to understand whether the four prophages are unique to B. anthracis and whether they produce active phages. Results More than 300 geographically and temporally divergent isolates of B. anthracis and its near neighbors were screened by PCR for the presence of specific DNA sequences from each prophage region. Every isolate of B. anthracis screened by PCR was found to produce all four phage-specific amplicons whereas none of the non-B. anthracis isolates, produced more than one phage-specific amplicon. Excision of prophages could be detected by a PCR based assay for attP sites on extra-chromosomal phage circles and for attB sites on phage-excised chromosomes. SYBR-green real-time PCR assays indicated that prophage excision occurs at very low frequencies (2 × 10-5 - 8 × 10-8/cell. Induction with mitomycin C increased the frequency of excision of one of the prophages by approximately 250 fold. All four prophages appear to be defective since, mitomycin C induced culture did not release any viable phage particle or lyse the cells or reveal any phage particle under electron microscopic examination. Conclusion The retention of all four putative prophage regions across all tested strains of B. anthracis is further evidence of the very recent emergence of this lineage and the prophage regions may be useful for differentiating the B. anthracis chromosome from that of its neighbors. All four prophages can excise at low frequencies, but are apparently defective in phage production.

  15. Basal ganglia calcification as a putative cause for cognitive decline

    Directory of Open Access Journals (Sweden)

    João Ricardo Mendes de Oliveira

    Full Text Available ABSTRACT Basal ganglia calcifications (BGC may be present in various medical conditions, such as infections, metabolic, psychiatric and neurological diseases, associated with different etiologies and clinical outcomes, including parkinsonism, psychosis, mood swings and dementia. A literature review was performed highlighting the main neuropsychological findings of BGC, with particular attention to clinical reports of cognitive decline. Neuroimaging studies combined with neuropsychological analysis show that some patients have shown progressive disturbances of selective attention, declarative memory and verbal perseveration. Therefore, the calcification process might represent a putative cause for dementia syndromes, suggesting a probable link among calcinosis, the aging process and eventually with neuronal death. The increasing number of reports available will foster a necessary discussion about cerebral calcinosis and its role in determining symptomatology in dementia patients

  16. Identification of putative and potential cross-reactive chickpea (Cicer arietinum) allergens through an in silico approach.

    Science.gov (United States)

    Kulkarni, Anuja; Ananthanarayan, Laxmi; Raman, Karthik

    2013-12-01

    Allergy has become a key cause of morbidity worldwide. Although many legumes (plants in the Fabaceae family) are healthy foods, they may have a number of allergenic proteins. A number of allergens have been identified and characterized in Fabaceae family, such as soybean and peanut, on the basis of biochemical and molecular biological approaches. However, our understanding of the allergens from chickpea (Cicer arietinum L.), belonging to this family, is very limited. In this study, we aimed to identify putative and cross-reactive allergens from Chickpea (C. arietinum) by means of in silico analysis of the chickpea protein sequences and allergens sequences from Fabaceae family. We retrieved known allergen sequences in Fabaceae family from the IUIS Allergen Nomenclature Database. We performed a protein BLAST (BLASTp) on these sequences to retrieve the similar sequences from chickpea. We further analyzed the retrieved chickpea sequences using a combination of in silico tools, to assess them for their allergenicity potential. Following this, we built structure models using FUGUE: Sequence-structure homology; these models generated by the recognition tool were viewed in Swiss-PDB viewer. Through this in silico approach, we identified seven novel putative allergens from chickpea proteome sequences on the basis of similarity of sequence, structure and physicochemical properties with the known reported legume allergens. Four out of seven putative allergens may also show cross reactivity with reported allergens since potential allergens had common sequence and structural features with the reported allergens. The in silico proteomic identification of the allergen proteins in chickpea provides a basis for future research on developing hypoallergenic foods containing chickpea. Such bioinformatics approaches, combined with experimental methodology, will help delineate an efficient and comprehensive approach to assess allergenicity and pave the way for a better understanding of

  17. Rapid Discrimination Among Putative Mechanistic Models of Biochemical Systems.

    Science.gov (United States)

    Lomnitz, Jason G; Savageau, Michael A

    2016-08-31

    An overarching goal in molecular biology is to gain an understanding of the mechanistic basis underlying biochemical systems. Success is critical if we are to predict effectively the outcome of drug treatments and the development of abnormal phenotypes. However, data from most experimental studies is typically noisy and sparse. This allows multiple potential mechanisms to account for experimental observations, and often devising experiments to test each is not feasible. Here, we introduce a novel strategy that discriminates among putative models based on their repertoire of qualitatively distinct phenotypes, without relying on knowledge of specific values for rate constants and binding constants. As an illustration, we apply this strategy to two synthetic gene circuits exhibiting anomalous behaviors. Our results show that the conventional models, based on their well-characterized components, cannot account for the experimental observations. We examine a total of 40 alternative hypotheses and show that only 5 have the potential to reproduce the experimental data, and one can do so with biologically relevant parameter values.

  18. Comparative genome analysis of 24 bovine-associated Staphylococcus isolates with special focus on the putative virulence genes

    Science.gov (United States)

    Åvall-Jääskeläinen, Silja; Paulin, Lars; Blom, Jochen

    2018-01-01

    Non-aureus staphylococci (NAS) are most commonly isolated from subclinical mastitis. Different NAS species may, however, have diverse effects on the inflammatory response in the udder. We determined the genome sequences of 20 staphylococcal isolates from clinical or subclinical bovine mastitis, belonging to the NAS species Staphylococcus agnetis, S. chromogenes, and S. simulans, and focused on the putative virulence factor genes present in the genomes. For comparison we used our previously published genome sequences of four S. aureus isolates from bovine mastitis. The pan-genome and core genomes of the non-aureus isolates were characterized. After that, putative virulence factor orthologues were searched in silico. We compared the presence of putative virulence factors in the NAS species and S. aureus and evaluated the potential association between bacterial genotype and type of mastitis (clinical vs. subclinical). The NAS isolates had much less virulence gene orthologues than the S. aureus isolates. One third of the virulence genes were detected only in S. aureus. About 100 virulence genes were present in all S. aureus isolates, compared to about 40 to 50 in each NAS isolate. S. simulans differed the most. Several of the virulence genes detected among NAS were harbored only by S. simulans, but it also lacked a number of genes present both in S. agnetis and S. chromogenes. The type of mastitis was not associated with any specific virulence gene profile. It seems that the virulence gene profiles or cumulative number of different virulence genes are not directly associated with the type of mastitis (clinical or subclinical), indicating that host derived factors such as the immune status play a pivotal role in the manifestation of mastitis. PMID:29610707

  19. Comparative genome analysis of 24 bovine-associated Staphylococcus isolates with special focus on the putative virulence genes

    Directory of Open Access Journals (Sweden)

    Silja Åvall-Jääskeläinen

    2018-03-01

    Full Text Available Non-aureus staphylococci (NAS are most commonly isolated from subclinical mastitis. Different NAS species may, however, have diverse effects on the inflammatory response in the udder. We determined the genome sequences of 20 staphylococcal isolates from clinical or subclinical bovine mastitis, belonging to the NAS species Staphylococcus agnetis, S. chromogenes, and S. simulans, and focused on the putative virulence factor genes present in the genomes. For comparison we used our previously published genome sequences of four S. aureus isolates from bovine mastitis. The pan-genome and core genomes of the non-aureus isolates were characterized. After that, putative virulence factor orthologues were searched in silico. We compared the presence of putative virulence factors in the NAS species and S. aureus and evaluated the potential association between bacterial genotype and type of mastitis (clinical vs. subclinical. The NAS isolates had much less virulence gene orthologues than the S. aureus isolates. One third of the virulence genes were detected only in S. aureus. About 100 virulence genes were present in all S. aureus isolates, compared to about 40 to 50 in each NAS isolate. S. simulans differed the most. Several of the virulence genes detected among NAS were harbored only by S. simulans, but it also lacked a number of genes present both in S. agnetis and S. chromogenes. The type of mastitis was not associated with any specific virulence gene profile. It seems that the virulence gene profiles or cumulative number of different virulence genes are not directly associated with the type of mastitis (clinical or subclinical, indicating that host derived factors such as the immune status play a pivotal role in the manifestation of mastitis.

  20. Evidence of ancient genome reduction in red algae (Rhodophyta).

    Science.gov (United States)

    Qiu, Huan; Price, Dana C; Yang, Eun Chan; Yoon, Hwan Su; Bhattacharya, Debashish

    2015-08-01

    Red algae (Rhodophyta) comprise a monophyletic eukaryotic lineage of ~6,500 species with a fossil record that extends back 1.2 billion years. A surprising aspect of red algal evolution is that sequenced genomes encode a relatively limited gene inventory (~5-10 thousand genes) when compared with other free-living algae or to other eukaryotes. This suggests that the common ancestor of red algae may have undergone extensive genome reduction, which can result from lineage specialization to a symbiotic or parasitic lifestyle or adaptation to an extreme or oligotrophic environment. We gathered genome and transcriptome data from a total of 14 red algal genera that represent the major branches of this phylum to study genome evolution in Rhodophyta. Analysis of orthologous gene gains and losses identifies two putative major phases of genome reduction: (i) in the stem lineage leading to all red algae resulting in the loss of major functions such as flagellae and basal bodies, the glycosyl-phosphatidylinositol anchor biosynthesis pathway, and the autophagy regulation pathway; and (ii) in the common ancestor of the extremophilic Cyanidiophytina. Red algal genomes are also characterized by the recruitment of hundreds of bacterial genes through horizontal gene transfer that have taken on multiple functions in shared pathways and have replaced eukaryotic gene homologs. Our results suggest that Rhodophyta may trace their origin to a gene depauperate ancestor. Unlike plants, it appears that a limited gene inventory is sufficient to support the diversification of a major eukaryote lineage that possesses sophisticated multicellular reproductive structures and an elaborate triphasic sexual cycle. © 2015 Phycological Society of America.

  1. Disparate subcellular location of putative sortase substrates in Clostridium difficile.

    Science.gov (United States)

    Peltier, Johann; Shaw, Helen A; Wren, Brendan W; Fairweather, Neil F

    2017-08-23

    Clostridium difficile is a gastrointestinal pathogen but how the bacterium colonises this niche is still little understood. Sortase enzymes covalently attach specific bacterial proteins to the peptidoglycan cell wall and are often involved in colonisation by pathogens. Here we show C. difficile proteins CD2537 and CD3392 are functional substrates of sortase SrtB. Through manipulation of the C-terminal regions of these proteins we show the SPKTG motif is essential for covalent attachment to the cell wall. Two additional putative substrates, CD0183 which contains an SPSTG motif, and CD2768 which contains an SPQTG motif, are not cleaved or anchored to the cell wall by sortase. Finally, using an in vivo asymmetric cleavage assay, we show that despite containing a conserved SPKTG motif, in the absence of SrtB these proteins are localised to disparate cellular compartments.

  2. Putative benefits of microalgal astaxanthin on exercise and human health

    Directory of Open Access Journals (Sweden)

    Marcelo P. Barros

    2011-04-01

    Full Text Available Astaxanthin (ASTA is a pinkish-orange carotenoid produced by microalgae, but also commonly found in shrimp, lobster and salmon, which accumulate ASTA from the aquatic food chain. Numerous studies have addressed the benefits of ASTA for human health, including the inhibition of LDL oxidation, UV-photoprotection and prophylaxis of bacterial stomach ulcers. ASTA is recognized as a powerful scavenger of reactive oxygen species (ROS, especially those involved in lipid peroxidation. Both aerobic and anaerobic exercise are closely related to overproduction of ROS in muscle tissue. Post-exercise inflammatory processes can even exacerbate the oxidative stress imposed by exercise. Thus, ASTA is suggested here as a putative nutritional alternative/coadjutant for antioxidant therapy to afford additional protection to muscle tissues against oxidative damage induced by exercise, as well as for an (overall integrative redox re-balance and general human health.

  3. Molecular detection of a putatively novel cyprinid herpesvirus in sichel (Pelecus cultratus) during a mass mortality event in Hungary.

    Science.gov (United States)

    Doszpoly, Andor; Papp, Melitta; Deákné, Petra P; Glávits, Róbert; Ursu, Krisztina; Dán, Ádám

    2015-05-01

    In the early summer of 2014, mass mortality of sichel (Pelecus cultratus) was observed in Lake Balaton, Hungary. Histological examination revealed degenerative changes within the tubular epithelium, mainly in the distal tubules and collecting ducts in the kidneys and multifocal vacuolisation in the brain stem and cerebellum. Routine molecular investigations showed the presence of the DNA of an unknown alloherpesvirus in some specimens. Subsequently, three genes of the putative herpesviral genome (DNA polymerase, terminase, and helicase) were amplified and partially sequenced. A phylogenetic tree reconstruction based on the concatenated sequence of these three conserved genes implied that the virus belongs to the genus Cyprinivirus within the family Alloherpesviridae. The sequences of the sichel herpesvirus differ markedly from those of the cypriniviruses CyHV-1, CyHV-2 and CyHV-3, putatively representing a fifth species in the genus.

  4. Identification of Putative Ortholog Gene Blocks Involved in Gestant and Lactating Mammary Gland Development: A Rodent Cross-Species Microarray Transcriptomics Approach

    Science.gov (United States)

    Rodríguez-Cruz, Maricela; Coral-Vázquez, Ramón M.; Hernández-Stengele, Gabriel; Sánchez, Raúl; Salazar, Emmanuel; Sanchez-Muñoz, Fausto; Encarnación-Guevara, Sergio; Ramírez-Salcedo, Jorge

    2013-01-01

    The mammary gland (MG) undergoes functional and metabolic changes during the transition from pregnancy to lactation, possibly by regulation of conserved genes. The objective was to elucidate orthologous genes, chromosome clusters and putative conserved transcriptional modules during MG development. We analyzed expression of 22,000 transcripts using murine microarrays and RNA samples of MG from virgin, pregnant, and lactating rats by cross-species hybridization. We identified 521 transcripts differentially expressed; upregulated in early (78%) and midpregnancy (89%) and early lactation (64%), but downregulated in mid-lactation (61%). Putative orthologous genes were identified. We mapped the altered genes to orthologous chromosomal locations in human and mouse. Eighteen sets of conserved genes associated with key cellular functions were revealed and conserved transcription factor binding site search entailed possible coregulation among all eight block sets of genes. This study demonstrates that the use of heterologous array hybridization for screening of orthologous gene expression from rat revealed sets of conserved genes arranged in chromosomal order implicated in signaling pathways and functional ontology. Results demonstrate the utilization power of comparative genomics and prove the feasibility of using rodent microarrays to identification of putative coexpressed orthologous genes involved in the control of human mammary gland development. PMID:24288657

  5. Localization and expression of putative circadian clock transcripts in the brain of the nudibranch Melibe leonina.

    Science.gov (United States)

    Duback, Victoria E; Sabrina Pankey, M; Thomas, Rachel I; Huyck, Taylor L; Mbarani, Izhar M; Bernier, Kyle R; Cook, Geoffrey M; O'Dowd, Colleen A; Newcomb, James M; Watson, Winsor H

    2018-09-01

    The nudibranch, Melibe leonina, expresses a circadian rhythm of locomotion, and we recently determined the sequences of multiple circadian clock transcripts that may play a role in controlling these daily patterns of behavior. In this study, we used these genomic data to help us: 1) identify putative clock neurons using fluorescent in situ hybridization (FISH); and 2) determine if there is a daily rhythm of expression of clock transcripts in the M. leonina brain, using quantitative PCR. FISH indicated the presence of the clock-related transcripts clock, period, and photoreceptive and non-photoreceptive cryptochrome (pcry and npcry, respectively) in two bilateral neurons in each cerebropleural ganglion and a group of <10 neurons in the anterolateral region of each pedal ganglion. Double-label experiments confirmed colocalization of all four clock transcripts with each other. Quantitative PCR demonstrated that the genes clock, period, pcry and npcry exhibited significant differences in expression levels over 24 h. These data suggest that the putative circadian clock network in M. leonina consists of a small number of identifiable neurons that express circadian genes with a daily rhythm. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition.

    LENUS (Irish Health Repository)

    Baeyens, A

    2002-12-02

    The chromosomal radiosensitivity of breast cancer patients with a known or putative genetic predisposition was investigated and compared to a group of healthy women. The chromosomal radiosensitivity was assessed with the G2 and the G0-micronucleus assay. For the G2 assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy (60)Co gamma-rays after 71 h incubation, and chromatid breaks were scored in 50 metaphases. For the micronucleus assay lymphocytes were exposed in vitro to 3.5 Gy (60)Co gamma-rays at a high dose rate or low dose rate. 70 h post-irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. The results demonstrated that the group of breast cancer patients with a known or putative genetic predisposition was on the average more radiosensitive than a population of healthy women, and this with the G2 as well as with the high dose rate and low dose rate micronucleus assay. With the G2 assay 43% of the patients were found to be radiosensitive. A higher proportion of the patients were radiosensitive with the micronucleus assay (45% with high dose rate and 61% with low dose rate). No correlation was found between the G2 and the G0-micronucleus chromosomal radiosensitivity. Out of the different subgroups considered, the group of the young breast cancer patients without family history showed the highest percentage of radiosensitive cases in the G2 (50%) as well as in the micronucleus assay (75-78%).

  7. Silencing of a putative immunophilin gene in the cattle tick Rhipicephalus (Boophilus microplus increases the infection rate of Babesia bovis in larval progeny

    Directory of Open Access Journals (Sweden)

    Knowles Donald P

    2009-11-01

    Full Text Available Abstract Background The cattle tick Rhipicephalus (Boophilus microplus is involved in the transmission of the protozoan Babesia bovis, the etiological agent of bovine babesiosis. Interactions between ticks and protozoa are poorly understood and the investigation of tick genes that affect tick fitness and protozoan infection can set the stage for dissecting the molecular interactions between the two species. Results In this study, RNA interference was used to silence R. microplus genes that had been previously shown to be up-regulated in response to B. bovis infection. The silencing of a putative immunophilin gene (Imnp in female ticks fed on a calf acutely infected with B. bovis decreased the hatching rate and survival of larval progeny. Interestingly, Imnp was up-regulated significantly in ovaries of R. microplus in response to B. bovis infection and its silencing in female ticks significantly increased the infection rate of the protozoan in larval progeny. The results also showed that the silencing of a putative Kunitz-type serine protease inhibitor (Spi gene and a putative lipocalin (Lpc gene decreased the fitness of R. microplus females, but had no significant effect on the infection rate of B. bovis in larval progeny. Conclusion The silencing of the Imnp, Spi or Lpc genes decreased the fitness of R. microplus females fed on a calf during acute B. bovis infection. The Imnp gene data suggest that this putative immunophilin gene is involved in the defense system of R. microplus against B. bovis and may play a role in controlling the protozoan infection in tick ovaries and larval progeny.

  8. La réputation de l'audit externe et les mécanismes de gouvernement d'entreprise: Interactions et effet sur la performance

    OpenAIRE

    Fodil Adjaoud; Chokri Mamoghli; Fatma Siala

    2007-01-01

    International audience; L'objectif de cette étude est d'analyser l'impact de la combinaison de la réputation de l'audit externe et des mécanismes internes de gouvernement d'entreprise sur la performance. Les tests menés sur un échantillon de 289 entreprises canadiennes sur trois ans supportent l'hypothèse de substitution proposée par la littérature. Nos résultats montrent qu'une meilleure réputation de l'audit pourrait ne plus constituer une prérogative aussi bien pour les actionnaires que po...

  9. Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

    OpenAIRE

    Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

    2016-01-01

    Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones, which include the xanthanolides. To date, the biogenesis of xanthanolides, especiallytheir downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes...

  10. Characterization of ERAS, a putative novel human oncogene, in skin and breast

    Energy Technology Data Exchange (ETDEWEB)

    Peña Avalos, B.L. de la

    2014-07-01

    Most human tumors have mutations in genes of the RAS small GTPase protein family. RAS works as a molecular switch for signaling pathways that modulate many aspects of cell behavior, including proliferation, differentiation, motility and death. Oncogenic mutations in RAS prevent GTP hydrolysis, locking RAS in a permanently active state, being the most common mutations in HRAS, KRAS and NRAS. The human RAS family consists of at least 36 different genes, many of which have been scarcely studied. One of these relatively unknown genes is ERAS (ES cell-expressed RAS), which is a constitutively active RAS protein, localized in chromosome X and expressed only in embryonic cells, being undetectable in adult tissues. New high throughput technologies have made it possible to screen complete cancer genomes for identification of mutations associated to cancer. Using the Sleeping Beauty (SB) transposon system, ERAS was identified as a putative novel oncogene in non-melanoma skin and breast cancers. The major aim of this project is to determine the general characteristics of ERAS as a putative novel human oncogene in skin and breast cells. Forced expression of ERAS results in drastic changes in cell shape, proliferation and motility. When ERAS is overexpressed in skin and breast human cells it is mainly localized in the cytoplasmic membrane. ERAS activates the phosphatidylinositol-3-OH kinase (PI3K) pathway but not the mitogen-activated protein kinase (MAPK) pathway. ERAS-expressing cells suffer spontaneous morphologic and phenotypic EMT-like changes, including cytoskeleton reorganization, vimentin and N-cadherin up-regulation and down-regulation of E-cadherin, which can be associated with increased malignancy, and invasive and metastatic potential. Our results suggest that inappropriate expression of ERAS lead to transformation of human cells. (Author)

  11. Differences in crystallization of two LinB variants from Sphingobium japonicum UT26

    Czech Academy of Sciences Publication Activity Database

    Degtjarik, O.; Chaloupková, R.; Řezáčová, Pavlína; Kutý, M.; Damborský, J.; Smatanová, I.K.

    2013-01-01

    Roč. 69, č. 3 (2013), s. 284-287 ISSN 1744-3091 Institutional support: RVO:68378050 Keywords : protein crystallization * dehalogenase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.568, year: 2013

  12. A strategy to discover new organizers identifies a putative heart organizer.

    Science.gov (United States)

    Anderson, Claire; Khan, Mohsin A F; Wong, Frances; Solovieva, Tatiana; Oliveira, Nidia M M; Baldock, Richard A; Tickle, Cheryll; Burt, Dave W; Stern, Claudio D

    2016-08-25

    Organizers are regions of the embryo that can both induce new fates and impart pattern on other regions. So far, surprisingly few organizers have been discovered, considering the number of patterned tissue types generated during development. This may be because their discovery has relied on transplantation and ablation experiments. Here we describe a new approach, using chick embryos, to discover organizers based on a common gene expression signature, and use it to uncover the anterior intestinal portal (AIP) endoderm as a putative heart organizer. We show that the AIP can induce cardiac identity from non-cardiac mesoderm and that it can pattern this by specifying ventricular and suppressing atrial regional identity. We also uncover some of the signals responsible. The method holds promise as a tool to discover other novel organizers acting during development.

  13. Phloem proteomics reveals new lipid-binding proteins with a putative role in lipid-mediated signaling

    Directory of Open Access Journals (Sweden)

    Allison Marie Barbaglia

    2016-04-01

    Full Text Available Global climate changes inversely affect our ability to grow the food required for an increasing world population. To combat future crop loss due to abiotic stress, we need to understand the signals responsible for changes in plant development and the resulting adaptations, especially the signaling molecules traveling long-distance through the plant phloem. Using a proteomics approach, we had identified several putative lipid-binding proteins in the phloem exudates. Simultaneously, we identified several complex lipids as well as jasmonates. These findings prompted us to propose that phloem (phospho- lipids could act as long-distance developmental signals in response to abiotic stress, and that they are released, sensed, and moved by phloem lipid-binding proteins (Benning et al., 2012. Indeed, the proteins we identified include lipases that could release a signaling lipid into the phloem, putative receptor components, and proteins that could mediate lipid-movement. To test this possible protein-based lipid-signaling pathway, three of the proteins, which could potentially act in a relay, are characterized here: (I a putative GDSL-motif lipase (II a PIG-P-like protein, with a possible receptor-like function; (III and PLAFP (phloem lipid-associated family protein, a predicted lipid-binding protein of unknown function. Here we show that all three proteins bind lipids, in particular phosphatidic acid (PtdOH, which is known to participate in intracellular stress signaling. Genes encoding these proteins are expressed in the vasculature, a prerequisite for phloem transport. Cellular localization studies show that the proteins are not retained in the endoplasmic reticulum but surround the cell in a spotted pattern that has been previously observed with receptors and plasmodesmatal proteins. Abiotic signals that induce the production of PtdOH also regulate the expression of GDSL-lipase and PLAFP, albeit in opposite patterns. Our findings suggest that while

  14. A Pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation.

    Science.gov (United States)

    Samin, Ghufrana; Pavlova, Martina; Arif, M Irfan; Postema, Christiaan P; Damborsky, Jiri; Janssen, Dick B

    2014-09-01

    1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. A putative peroxidase cDNA from turnip and analysis of the encoded protein sequence.

    Science.gov (United States)

    Romero-Gómez, S; Duarte-Vázquez, M A; García-Almendárez, B E; Mayorga-Martínez, L; Cervantes-Avilés, O; Regalado, C

    2008-12-01

    A putative peroxidase cDNA was isolated from turnip roots (Brassica napus L. var. purple top white globe) by reverse transcriptase-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Total RNA extracted from mature turnip roots was used as a template for RT-PCR, using a degenerated primer designed to amplify the highly conserved distal motif of plant peroxidases. The resulting partial sequence was used to design the rest of the specific primers for 5' and 3' RACE. Two cDNA fragments were purified, sequenced, and aligned with the partial sequence from RT-PCR, and a complete overlapping sequence was obtained and labeled as BbPA (Genbank Accession No. AY423440, named as podC). The full length cDNA is 1167bp long and contains a 1077bp open reading frame (ORF) encoding a 358 deduced amino acid peroxidase polypeptide. The putative peroxidase (BnPA) showed a calculated Mr of 34kDa, and isoelectric point (pI) of 4.5, with no significant identity with other reported turnip peroxidases. Sequence alignment showed that only three peroxidases have a significant identity with BnPA namely AtP29a (84%), and AtPA2 (81%) from Arabidopsis thaliana, and HRPA2 (82%) from horseradish (Armoracia rusticana). Work is in progress to clone this gene into an adequate host to study the specific role and possible biotechnological applications of this alternative peroxidase source.

  16. Complete genome sequence of two tomato-infecting begomoviruses in Venezuela: evidence of a putative novel species and a novel recombinant strain.

    Science.gov (United States)

    Romay, Gustavo; Chirinos, Dorys T; Geraud-Pouey, Francis; Gillis, Annika; Mahillon, Jacques; Bragard, Claude

    2018-02-01

    At least six begomovirus species have been reported infecting tomato in Venezuela. In this study the complete genomes of two tomato-infecting begomovirus isolates (referred to as Trujillo-427 and Zulia-1084) were cloned and sequenced. Both isolates showed the typical genome organization of New World bipartite begomoviruses, with DNA-A genomic components displaying 88.8% and 90.3% similarity with established begomoviruses, for isolates Trujillo-427 and Zulia-1084, respectively. In accordance to the guidelines for begomovirus species demarcation, the Trujillo-427 isolate represents a putative new species and the name "Tomato wrinkled mosaic virus" is proposed. Meanwhile, Zulia-1084 represents a putative new strain classifiable within species Tomato chlorotic leaf distortion virus, for which a recombinant origin is suggested.

  17. Comparison of formation of reactive conformers for the SN2 displacements by CH3CO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} in water and by Asp124-CO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document} in a haloalkane dehalogenase

    Science.gov (United States)

    Hur, Sun; Kahn, Kalju; Bruice, Thomas C.

    2003-01-01

    The SN2 displacement of Cl− from 1,2-dichloroethane by acetate (CH3CO\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}{\\mathrm{_{2}^{-}}}\\end{equation*}\\end{document}) in water and by the carboxylate of the active site aspartate in the haloalkane dehalogenase of Xanthobacter autothropicus have been compared by using molecular dynamics simulations. In aqueous solution, six families of contact-pair structures (I–VI) were identified, and their relative concentrations and dissociation rate constants were determined. The near attack conformers (NACs) required for the SN2 displacement reaction are members of the IV (CH3COO−⋅ ⋅ ⋅CH2(Cl)CH2Cl) family and are formed in the sequence II→III→IV→NAC. The NAC subclass is defined by the —COO−⋅ ⋅ ⋅C—Cl contact distance of ≤3.41 Å and the —COO−⋅ ⋅ ⋅C—Cl angle of 157–180°. The mole percentage of NACs is 0.16%, based on the 1 M standard state. This result may be compared with 13.4 mole percentage of NACs in the Michaelis complex in the enzyme. It follows that NAC formation in the enzyme is favored by 2.6 kcal/mol. Because reaction coordinates from S to TS, both in water and in the enzyme, pass via NAC (i.e., S → NAC → TS), the reduction in the S → NAC barrier by 2.6 kcal/mol accounts for ≈25% of the reduction of total barrier in the S → TS (10.7 kcal/mol). The remaining 75% of the advantage of the enzymatic reaction revolves around the efficiency of NAC → TS step. This process, based on previous studies, is discussed briefly. PMID:12610210

  18. VISLISI trial, a prospective clinical study allowing identification of a new metalloprotease and putative virulence factor from Staphylococcus lugdunensis.

    Science.gov (United States)

    Argemi, X; Prévost, G; Riegel, P; Keller, D; Meyer, N; Baldeyrou, M; Douiri, N; Lefebvre, N; Meghit, K; Ronde Oustau, C; Christmann, D; Cianférani, S; Strub, J M; Hansmann, Y

    2017-05-01

    Staphylococcus lugdunensis is a coagulase-negative staphylococcus that displays an unusually high virulence rate close to that of Staphylococcus aureus. It also shares phenotypic properties with S. aureus and several studies found putative virulence factors. The objective of the study was to describe the clinical manifestations of S. lugdunensis infections and investigate putative virulence factors. We conducted a prospective study from November 2013 to March 2016 at the University Hospital of Strasbourg. Putative virulence factors were investigated by clumping factor detection, screening for proteolytic activity, and sequence analysis using tandem nano-liquid chromatography-mass spectrometry. In total, 347 positive samples for S. lugdunensis were collected, of which 129 (37.2%) were from confirmed cases of S. lugdunensis infection. Eighty-one of these 129 patients were included in the study. Bone and prosthetic joints (PJI) were the most frequent sites of infection (n=28; 34.6%) followed by skin and soft tissues (n=23; 28.4%). We identified and purified a novel protease secreted by 50 samples (61.7%), most frequently associated with samples from deep infections and PJI (pr 0.97 and pr 0.91, respectively). Protease peptide sequencing by nano-liquid chromatography-mass spectrometry revealed a novel protease bearing 62.42% identity with ShpI, a metalloprotease secreted by Staphylococcus hyicus. This study confirms the pathogenicity of S. lugdunensis, particularly in bone and PJI. We also identified a novel metalloprotease called lugdulysin that may contribute to virulence. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  19. In silico Prediction, in vitro Antibacterial Spectrum, and Physicochemical Properties of a Putative Bacteriocin Produced by Lactobacillus rhamnosus Strain L156.4

    Directory of Open Access Journals (Sweden)

    Letícia de C. Oliveira

    2017-05-01

    Full Text Available A bacteriocinogenic Lactobacillus rhamnosus L156.4 strain isolated from the feces of NIH mice was identified by 16S rRNA gene sequencing and MALDI-TOF mass spectrometry. The entire genome was sequenced using Illumina, annotated in the PGAAP, and RAST servers, and deposited. Conserved genes associated with bacteriocin synthesis were predicted using BAGEL3, leading to the identification of an open reading frame (ORF that shows homology with the L. rhamnosus GG (ATCC 53103 prebacteriocin gene. The encoded protein contains a conserved protein motif associated a structural gene of the Enterocin A superfamily. We found ORFs related to the prebacteriocin, immunity protein, ABC transporter proteins, and regulatory genes with 100% identity to those of L. rhamnosus HN001. In this study, we provide evidence of a putative bacteriocin produced by L. rhamnosus L156.4 that was further confirmed by in vitro assays. The antibacterial activity of the substances produced by this strain was evaluated using the deferred agar-spot and spot-on-the lawn assays, and a wide antimicrobial activity spectrum against human and foodborne pathogens was observed. The physicochemical characterization of the putative bacteriocin indicated that it was sensitive to proteolytic enzymes, heat stable and maintained its antibacterial activity in a pH ranging from 3 to 9. The activity against Lactobacillus fermentum, which was used as an indicator strain, was detected during bacterial logarithmic growth phase, and a positive correlation was confirmed between bacterial growth and production of the putative bacteriocin. After a partial purification from cell-free supernatant by salt precipitation, the putative bacteriocin migrated as a diffuse band of approximately 1.0–3.0 kDa by SDS-PAGE. Additional studies are being conducted to explore its use in the food industry for controlling bacterial growth and for probiotic applications.

  20. In silico Prediction, in vitro Antibacterial Spectrum, and Physicochemical Properties of a Putative Bacteriocin Produced by Lactobacillus rhamnosus Strain L156.4

    Science.gov (United States)

    Oliveira, Letícia de C.; Silveira, Aline M. M.; Monteiro, Andréa de S.; dos Santos, Vera L.; Nicoli, Jacques R.; Azevedo, Vasco A. de C.; Soares, Siomar de C.; Dias-Souza, Marcus V.; Nardi, Regina M. D.

    2017-01-01

    A bacteriocinogenic Lactobacillus rhamnosus L156.4 strain isolated from the feces of NIH mice was identified by 16S rRNA gene sequencing and MALDI-TOF mass spectrometry. The entire genome was sequenced using Illumina, annotated in the PGAAP, and RAST servers, and deposited. Conserved genes associated with bacteriocin synthesis were predicted using BAGEL3, leading to the identification of an open reading frame (ORF) that shows homology with the L. rhamnosus GG (ATCC 53103) prebacteriocin gene. The encoded protein contains a conserved protein motif associated a structural gene of the Enterocin A superfamily. We found ORFs related to the prebacteriocin, immunity protein, ABC transporter proteins, and regulatory genes with 100% identity to those of L. rhamnosus HN001. In this study, we provide evidence of a putative bacteriocin produced by L. rhamnosus L156.4 that was further confirmed by in vitro assays. The antibacterial activity of the substances produced by this strain was evaluated using the deferred agar-spot and spot-on-the lawn assays, and a wide antimicrobial activity spectrum against human and foodborne pathogens was observed. The physicochemical characterization of the putative bacteriocin indicated that it was sensitive to proteolytic enzymes, heat stable and maintained its antibacterial activity in a pH ranging from 3 to 9. The activity against Lactobacillus fermentum, which was used as an indicator strain, was detected during bacterial logarithmic growth phase, and a positive correlation was confirmed between bacterial growth and production of the putative bacteriocin. After a partial purification from cell-free supernatant by salt precipitation, the putative bacteriocin migrated as a diffuse band of approximately 1.0–3.0 kDa by SDS-PAGE. Additional studies are being conducted to explore its use in the food industry for controlling bacterial growth and for probiotic applications. PMID:28579977

  1. Putative radioresistant bacterial isolate from sewage water

    International Nuclear Information System (INIS)

    Ang, April; Chua, Patricia; Perez, Kristine; Rey, April; Rivor Kristel; San Pablo, Czarina; Santos, Ernestine

    2001-01-01

    Sewage water was collected from a stagnant body of water in Balara, Quezon City. approximately 150 ml was aseptically transferred into eight Erlenmeyer flasks. Seven flasks were then subjected to different doses of radiation at the 60 Co irradiation facility, PNRI (Philippine Nuclear Research Institute) which are as follows: 0.01 kGy, 0.1 kGy, 0.5 kGy, 1 kGy, 5 kGy, 10 kGy, and 15 kGy. The remaining flask was used as the control. After irradiation, all the different treatments were subjected to colony count at the culture collection laboratory, NSRI. Results showed that the colonies from sewage water treatments irradiated at 0.01 kGy (treatment A), 0.10 kGy (treatment B), and 0.50 kGy (treatment C) exhibited a decreasing trend with colony counts 4.60 x 10 3 CFU/ml, and 1.30 x 10 3 CFU/ml, and 26 CFU/ml, respectively. Contrastingly, at 1 kGy (treatment D), high colony count of 2.95 x 10 3 CFU/ml was observed which is even higher compared to the control (1.02 x 10 3 CFU/ml). Treatment E that was irradiated at 5 kGy manifested low survival rate (25 CFU/ml) indicating the presence of few putative intermediate radioresistant bacteria. Radiation dose treatments higher than 5 kGy (i.e., 10 kGy and 15 kGy) exhibited no bacterial survival. (Author)

  2. Putative radioresistant bacterial isolate from sewage water

    Energy Technology Data Exchange (ETDEWEB)

    Ang, April; Chua, Patricia; Perez, Kristine; Rey, April; Kristel, Rivor; San Pablo, Czarina; Santos, Ernestine

    2001-01-29

    Sewage water was collected from a stagnant body of water in Balara, Quezon City. approximately 150 ml was aseptically transferred into eight Erlenmeyer flasks. Seven flasks were then subjected to different doses of radiation at the {sup 60}Co irradiation facility, PNRI (Philippine Nuclear Research Institute) which are as follows: 0.01 kGy, 0.1 kGy, 0.5 kGy, 1 kGy, 5 kGy, 10 kGy, and 15 kGy. The remaining flask was used as the control. After irradiation, all the different treatments were subjected to colony count at the culture collection laboratory, NSRI. Results showed that the colonies from sewage water treatments irradiated at 0.01 kGy (treatment A), 0.10 kGy (treatment B), and 0.50 kGy (treatment C) exhibited a decreasing trend with colony counts 4.60 x 10{sup 3} CFU/ml, and 1.30 x 10{sup 3} CFU/ml, and 26 CFU/ml, respectively. Contrastingly, at 1 kGy (treatment D), high colony count of 2.95 x 10{sup 3} CFU/ml was observed which is even higher compared to the control (1.02 x 10{sup 3} CFU/ml). Treatment E that was irradiated at 5 kGy manifested low survival rate (25 CFU/ml) indicating the presence of few putative intermediate radioresistant bacteria. Radiation dose treatments higher than 5 kGy (i.e., 10 kGy and 15 kGy) exhibited no bacterial survival. (Author)

  3. Anaerobic microbial dehalogenation of organohalides-state of the art and remediation strategies.

    Science.gov (United States)

    Nijenhuis, Ivonne; Kuntze, Kevin

    2016-04-01

    Contamination and remediation of groundwater with halogenated organics and understanding of involved microbial reactions still poses a challenge. Over the last years, research in anaerobic microbial dehalogenation has advanced in many aspects providing information about the reaction, physiology of microorganisms as well as approaches to investigate the activity of microorganisms in situ. Recently published crystal structures of reductive dehalogenases (Rdh), heterologous expression systems and advanced analytical, proteomic and stable isotope approaches allow addressing the overall reaction and specific enzymes as well as co-factors involved during anaerobic microbial dehalogenation. In addition to Dehalococcoides spp., Dehalobacter and Dehalogenimonas strains have been recognized as important and versatile organohalide respirers. Together, these provide perspectives for integrated concepts allowing to improve and monitor in situ biodegradation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Regulation of Arabidopsis Early Anther Development by Putative Cell-Cell Signaling Molecules and Transcriptional Regulators

    Institute of Scientific and Technical Information of China (English)

    Yu-Jin Sun; Carey LH Hord; Chang-Bin Chen; Hong Ma

    2007-01-01

    Anther development in flowering plants involves the formation of several cell types, including the tapetal and pollen mother cells. The use of genetic and molecular tools has led to the identification and characterization of genes that are critical for normal cell division and differentiation in Arabidopsis early anther development. We review here several recent studies on these genes, including the demonstration that the putative receptor protein kinases BAM1 and BAM2 together play essential roles in the control of early cell division and differentiation. In addition, we discuss the hypothesis that BAM1/2 may form a positive-negative feedback regulatory loop with a previously identified key regulator, SPOROCYTELESS (also called NOZZLE),to control the balance between sporogenous and somatic cell types in the anther. Furthermore, we summarize the isolation and functional analysis of the DYSFUNCTIONAL TAPETUM1 (DYT1) gene in promoting proper tapetal cell differentiation. Our finding that DYT1 encodes a putative transcription factor of the bHLH family, as well as relevant expression analyses, strongly supports a model that DYT1 serves as a critical link between upstream factors and downstream target genes that are critical for normal tapetum development and function. These studies, together with other recently published works, indicate that cell-cell communication and transcriptional control are key processes essential for cell fate specification in anther development.

  5. Reduction and restoration of culturability of beer-stressed and low-temperature-stressed Lactobacillus acetotolerans strain 2011-8.

    Science.gov (United States)

    Deng, Yang; Liu, Junyan; Li, Lin; Fang, Huijing; Tu, Jingxia; Li, Bing; Liu, Jing; Li, Huiping; Xu, Zhenbo

    2015-08-03

    Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria, regardless of beer type, and therefore pose significant problems for the brewing industry. The aim of this study was to investigate the viable, but putatively non-culturable (VPNC) state of the hard-to-culture beer-spoilage species, Lactobacillus acetotolerans. Upon prolonged contact with degassed beer, L. acetotolerans was found to show decreased culturability. After 17 subcultures in beer, 100-μL aliquots of the culture were no longer culturable on MRS agar until 14 days of incubation despite the presence of 10(5) viable cells, indicating that a large population of cells entered into a VPNC state. Furthermore, a significant reduction or even putative loss of culturability, but maintenance of viability, of L. acetotolerans could also be induced by storing the strain at 0 °C for 105 days. Adding catalase at a concentration of 1000 U/plate enabled the VPNC cells, both induced by beer subculture treatment and cold treatment, to regain culturability with a resuscitation time of 4 days and 3 days, respectively. Scanning electron microscopy results demonstrated that cells decreased in size and gradually changed morphology from short rods to coccoids when they entered the VPNC state. It was concluded that the difficulty in culturing the spoilage bacterium from brewery environments could be partly attributed the hard-to-culture or the viable, but non-culturable characteristic of this organism. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Prevalence of Clinical Periodontitis and Putative Periodontal Pathogens among South Indian Pregnant Women

    Directory of Open Access Journals (Sweden)

    Chaitanya Tellapragada

    2014-01-01

    Full Text Available In view of recent understanding of the association of periodontal infections and adverse pregnancy outcomes, the present investigation was undertaken to study the periodontal infections among 390 asymptomatic pregnant women and to find an association of bacterial etiologies with the disease. Prevalence of gingivitis was 38% and clinical periodontitis was 10% among the study population. Subgingival plaque specimens were subjected to multiplex PCR targeting ten putative periodontopathogenic bacteria. Among the periodontitis group, high detection rates of Porphyromonas gingivalis (56%, Prevotella nigrescens (44%, Treponema denticola (32%, and Prevotella intermedius (24% were noted along with significant association with the disease (P<0.05.

  7. Purification, crystallization and preliminary X-ray diffraction analysis of the putative ABC transporter ATP-binding protein from Thermotoga maritima

    International Nuclear Information System (INIS)

    Ethayathulla, Abdul S.; Bessho, Yoshitaka; Shinkai, Akeo; Padmanabhan, Balasundaram; Singh, Tej P.; Kaur, Punit; Yokoyama, Shigeyuki

    2008-01-01

    The putative ABC transporter ATP-binding protein TM0222 from T. maritima was cloned, overproduced, purified and crystallized. A complete MAD diffraction data set has been collected to 2.3 Å resolution. Adenosine triphosphate (ATP) binding cassette transporters (ABC transporters) are ATP hydrolysis-dependent transmembrane transporters. Here, the overproduction, purification and crystallization of the putative ABC transporter ATP-binding protein TM0222 from Thermotoga maritima are reported. The protein was crystallized in the hexagonal space group P6 4 22, with unit-cell parameters a = b = 148.49, c = 106.96 Å, γ = 120.0°. Assuming the presence of two molecules in the asymmetric unit, the calculated V M is 2.84 Å 3 Da −1 , which corresponds to a solvent content of 56.6%. A three-wavelength MAD data set was collected to 2.3 Å resolution from SeMet-substituted TM0222 crystals. Data sets were collected on the BL38B1 beamline at SPring-8, Japan

  8. One pot 'click' reactions : tandem enantioselective biocatalytic epoxide ring opening and [3+2] azide alkyne cycloaddition

    NARCIS (Netherlands)

    Campbell-Verduyn, Lachlan S.; Szymanski, Wiktor; Postema, Christiaan P.; Dierckx, Rudi A.; Elsinga, Philip H.; Janssen, Dick B.; Feringa, Ben L.

    2010-01-01

    Halohydrin dehalogenase (HheC) can perform enantioselective azidolysis of aromatic epoxides to 1,2-azido alcohols which are subsequently ligated to alkynes producing chiral hydroxy triazoles in a one-pot procedure with excellent enantiomeric excess.

  9. Comparison of 454-ESTs from Huperzia serrata and Phlegmariurus carinatus reveals putative genes involved in lycopodium alkaloid biosynthesis and developmental regulation

    Directory of Open Access Journals (Sweden)

    Steinmetz André

    2010-09-01

    Full Text Available Abstract Background Plants of the Huperziaceae family, which comprise the two genera Huperzia and Phlegmariurus, produce various types of lycopodium alkaloids that are used to treat a number of human ailments, such as contusions, swellings and strains. Huperzine A, which belongs to the lycodine type of lycopodium alkaloids, has been used as an anti-Alzheimer's disease drug candidate. Despite their medical importance, little genomic or transcriptomic data are available for the members of this family. We used massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform to generate a substantial EST dataset for Huperzia serrata (H. serrata and Phlegmariurus carinatus (P. carinatus as representative members of the Huperzia and Phlegmariurus genera, respectively. H. serrata and P. carinatus are important plants for research on the biosynthesis of lycopodium alkaloids. We focused on gene discovery in the areas of bioactive compound biosynthesis and transcriptional regulation as well as genetic marker detection in these species. Results For H. serrata, 36,763 unique putative transcripts were generated from 140,930 reads totaling over 57,028,559 base pairs; for P. carinatus, 31,812 unique putative transcripts were generated from 79,920 reads totaling over 30,498,684 base pairs. Using BLASTX searches of public databases, 16,274 (44.3% unique putative transcripts from H. serrata and 14,070 (44.2% from P. carinatus were assigned to at least one protein. Gene Ontology (GO and Kyoto Encyclopedia of Genes and Genomes (KEGG orthology annotations revealed that the functions of the unique putative transcripts from these two species cover a similarly broad set of molecular functions, biological processes and biochemical pathways. In particular, a total of 20 H. serrata candidate cytochrome P450 genes, which are more abundant in leaves than in roots and might be involved in lycopodium alkaloid biosynthesis, were found based on the comparison of H

  10. PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

    Science.gov (United States)

    THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

  11. Report on the development of putative functional SSR and SNP markers in passion fruits.

    Science.gov (United States)

    da Costa, Zirlane Portugal; Munhoz, Carla de Freitas; Vieira, Maria Lucia Carneiro

    2017-09-06

    Passionflowers Passiflora edulis and Passiflora alata are diploid, outcrossing and understudied fruit bearing species. In Brazil, passion fruit cultivation began relatively recently and has earned the country an outstanding position as the world's top producer of passion fruit. The fruit's main economic value lies in the production of juice, an essential exotic ingredient in juice blends. Currently, crop improvement strategies, including those for underexploited tropical species, tend to incorporate molecular genetic approaches. In this study, we examined a set of P. edulis transcripts expressed in response to infection by Xanthomonas axonopodis, (the passion fruit's main bacterial pathogen that attacks the vines), aiming at the development of putative functional markers, i.e. SSRs (simple sequence repeats) and SNPs (single nucleotide polymorphisms). A total of 210 microsatellites were found in 998 sequences, and trinucleotide repeats were found to be the most frequent (31.4%). Of the sequences selected for designing primers, 80.9% could be used to develop SSR markers, and 60.6% SNP markers for P. alata. SNPs were all biallelic and found within 15 gene fragments of P. alata. Overall, gene fragments generated 10,003 bp. SNP frequency was estimated as one SNP every 294 bp. Polymorphism rates revealed by SSR and SNP loci were 29.4 and 53.6%, respectively. Passiflora edulis transcripts were useful for the development of putative functional markers for P. alata, suggesting a certain level of sequence conservation between these cultivated species. The markers developed herein could be used for genetic mapping purposes and also in diversity studies.

  12. The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12- and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study.

    Directory of Open Access Journals (Sweden)

    Sandro Roselli

    Full Text Available Chloromethane (CH3Cl is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD, as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2. In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex, conversion of

  13. Reduction and Methyl Transfer Kinetics of the Alpha Subunit from Acetyl-Coenzyme A Synthase

    Energy Technology Data Exchange (ETDEWEB)

    Xiangshi Tan; Christopher Sewell; Qingwu Yang; Paul A. Lindahl

    2003-01-15

    OAK-B135 Stopped-flow was used to evaluate the methylation and reduction kinetics of the isolated alpha subunit of acetyl-Coenzyme A synthase from Moorella thermoacetica. This catalytically active subunit contains a novel Ni-X-Fe4S4 cluster and a putative unidentified n =2 redox site called D. The D-site must be reduced for a methyl group to transfer from a corrinoid-iron-sulfur protein, a key step in the catalytic synthesis of acetyl-CoA. The Fe4S4 component of this cluster is also redox active, raising the possibility that it is the D-site or a portion thereof. Results presented demonstrate that the D-site reduces far faster than the Fe4S4 component, effectively eliminating this possibility. Rather, this component may alter catalytically important properties of the Ni center. The D-site is reduced through a pathway that probably does not involve the Fe4S4 component of this active-site cluster.

  14. Effects of activated carbon on reductive dechlorination of PCBs by organohalide respiring bacteria indigenous to sediments.

    Science.gov (United States)

    Kjellerup, B V; Naff, C; Edwards, S J; Ghosh, U; Baker, J E; Sowers, K R

    2014-04-01

    Polychlorinated biphenyls (PCBs) have accumulated in aquatic sediments due to their inherent chemical stability and their presence poses a risk due to their potential toxicity in humans and animals. Granular activated carbon (GAC) has been applied to PCB contaminated sediment sites to reduce the aqueous concentration by sequestration thus reducing the PCB exposure and toxicity to both benthic and aquatic organisms. However, it is not known how the reduction of PCB bioavailability by adsorption to GAC affects bacterial transformation of PCBs by indigenous organohalide respiring bacteria. In this study, the impact of GAC on anaerobic dechlorination by putative organohalide respiring bacteria indigenous to sediment from Baltimore Harbor was examined. It was shown that the average Cl/biphenyl after dehalogenation of Aroclor 1260 was similar between treatments with and without GAC amendment. However, GAC caused a substantial shift in the congener distribution whereby a smaller fraction of highly chlorinated congeners was more extensively dechlorinated to mono- through tri-chlorinated congeners compared to the formation of tri- through penta-chlorinated congeners in unamended sediment. The results combined with comparative sequence analysis of 16S rRNA gene sequences suggest that GAC caused a community shift to putative organohalide respiring phylotypes that coincided with more extensive dechlorination of ortho and unflanked chlorines. This shift in activity by GAC shown here for the first time has the potential to promote greater degradation in situ by promoting accumulation of less chlorinated congeners that are generally more susceptible to complete mineralization by aerobic PCB degrading bacteria. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Elucidating the Molecular Basis and Regulation of Chromium (VI) Reduction by Shewanella oneidensis MR-1 Using Biochemical, Genomic, and Proteomic Approaches

    Energy Technology Data Exchange (ETDEWEB)

    Hettich, Robert L.

    2006-10-30

    Although microbial metal reduction has been investigated intensively from physiological and biochemical perspectives, little is known about the genetic basis and regulatory mechanisms underlying the ability of certain bacteria to transform, detoxify, or immobilize a wide array of heavy metals contaminating DOE-relevant environments. The major goal of this work is to elucidate the molecular components comprising the chromium(VI) response pathway, with an emphasis on components involved in Cr(VI) detoxification and the enzyme complex catalyzing the terminal step in Cr(VI) reduction by Shewanella oneidensis MR-1. We have identified and characterized (in the case of DNA-binding response regulator [SO2426] and a putative azoreductase [SO3585]) the genes and gene products involved in the molecular response of MR-1 to chromium(VI) stress using whole-genome sequence information for MR-1 and recently developed proteomic technology, in particular liquid chromatographymass spectrometry (LC-MS), in conjunction with conventional protein purification and characterization techniques. The proteome datasets were integrated with information from whole-genome expression arrays for S. oneidensis MR-1 (as illustrated in Figure 1). The genes and their encoded products identified in this study are of value in understanding metal reduction and bacterial resistance to metal toxicity and in developing effective metal immobilization strategies.

  16. The V-ATPase a2-subunit as a putative endosomal pH-sensor.

    Science.gov (United States)

    Marshansky, V

    2007-11-01

    V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

  17. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    International Nuclear Information System (INIS)

    Fernández-Sainz, I.J.; Largo, E.; Gladue, D.P.; Fletcher, P.; O’Donnell, V.; Holinka, L.G.; Carey, L.B.; Lu, X.; Nieva, J.L.; Borca, M.V.

    2014-01-01

    E2, along with E rns and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, 818 CPIGWTGVIEC 828 , containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP 818 CPIGWTGVIEC 828 indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion

  18. Effect of specific amino acid substitutions in the putative fusion peptide of structural glycoprotein E2 on Classical Swine Fever Virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Fernández-Sainz, I.J. [Plum Island Animal Disease Center, ARS, USDA (United States); Largo, E. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Gladue, D.P.; Fletcher, P. [Plum Island Animal Disease Center, ARS, USDA (United States); O’Donnell, V. [Plum Island Animal Disease Center, ARS, USDA (United States); Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Holinka, L.G. [Plum Island Animal Disease Center, ARS, USDA (United States); Carey, L.B. [Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), E-08003 Barcelona (Spain); Lu, X. [Plum Island Animal Disease Center, DHS, Greenport, NY 11944 (United States); Nieva, J.L. [Biophysics Unit (CSIC-UPV/EHU), Department of Biochemistry and Molecular Biology, University of the Basque Country (UPV/EHU), P.O. Box 644, 48080 Bilbao (Spain); Borca, M.V., E-mail: manuel.borca@ars.usda.gov [Plum Island Animal Disease Center, ARS, USDA (United States)

    2014-05-15

    E2, along with E{sup rns} and E1, is an envelope glycoprotein of Classical Swine Fever Virus (CSFV). E2 is involved in several virus functions: cell attachment, host range susceptibility and virulence in natural hosts. Here we evaluate the role of a specific E2 region, {sup 818}CPIGWTGVIEC{sup 828}, containing a putative fusion peptide (FP) sequence. Reverse genetics utilizing a full-length infectious clone of the highly virulent CSFV strain Brescia (BICv) was used to evaluate how individual amino acid substitutions within this region of E2 may affect replication of BICv. A synthetic peptide representing the complete E2 FP amino acid sequence adopted a β-type extended conformation in membrane mimetics, penetrated into model membranes, and perturbed lipid bilayer integrity in vitro. Similar peptides harboring amino acid substitutions adopted comparable conformations but exhibited different membrane activities. Therefore, a preliminary characterization of the putative FP {sup 818}CPIGWTGVIEC{sup 828} indicates a membrane fusion activity and a critical role in virus replication. - Highlights: • A putative fusion peptide (FP) region in CSFV E2 protein was shown to be critical for virus growth. • Synthetic FPs were shown to efficiently penetrate into lipid membranes using an in vitro model. • Individual residues in the FP affecting virus replication were identified by reverse genetics. • The same FP residues are also responsible for mediating membrane fusion.

  19. Evaluating iodide recycling inhibition as a novel molecular initiating event for thyroid axis disruption

    Science.gov (United States)

    The enzyme iodotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. Loss of function or chemical inhibition of IYD reduces available iodide for thyroid hormone synthesis, which leads to hormone insufficiency...

  20. Serotonin(4) (5-HT(4)) receptor agonists are putative antidepressants with a rapid onset of action

    DEFF Research Database (Denmark)

    Lucas, Guillaume; Rymar, Vladimir V; Du, Jenny

    2007-01-01

    parameters considered to be key markers of antidepressant action, but that are observed only after 2-3 week treatments with classical molecules: desensitization of 5-HT(1A) autoreceptors, increased tonus on hippocampal postsynaptic 5-HT(1A) receptors, and enhanced phosphorylation of the CREB protein...... intake consecutive to a chronic mild stress. These findings point out 5-HT(4) receptor agonists as a putative class of antidepressants with a rapid onset of action. Udgivelsesdato: 2007-Sep-6...

  1. EST mining identifies proteins putatively secreted by the anthracnose pathogen Colletotrichum truncatum

    Directory of Open Access Journals (Sweden)

    Vandenberg Albert

    2011-06-01

    Full Text Available Abstract Background Colletotrichum truncatum is a haploid, hemibiotrophic, ascomycete fungal pathogen that causes anthracnose disease on many economically important leguminous crops. This pathogen exploits sequential biotrophic- and necrotrophic- infection strategies to colonize the host. Transition from biotrophy to a destructive necrotrophic phase called the biotrophy-necrotrophy switch is critical in symptom development. C. truncatum likely secretes an arsenal of proteins that are implicated in maintaining a compatible interaction with its host. Some of them might be transition specific. Results A directional cDNA library was constructed from mRNA isolated from infected Lens culinaris leaflet tissues displaying the biotrophy-necrotrophy switch of C. truncatum and 5000 expressed sequence tags (ESTs with an average read of > 600 bp from the 5-prime end were generated. Nearly 39% of the ESTs were predicted to encode proteins of fungal origin and among these, 162 ESTs were predicted to contain N-terminal signal peptides (SPs in their deduced open reading frames (ORFs. The 162 sequences could be assembled into 122 tentative unigenes comprising 32 contigs and 90 singletons. Sequence analyses of unigenes revealed four potential groups: hydrolases, cell envelope associated proteins (CEAPs, candidate effectors and other proteins. Eleven candidate effector genes were identified based on features common to characterized fungal effectors, i.e. they encode small, soluble (lack of transmembrane domain, cysteine-rich proteins with a putative SP. For a selected subset of CEAPs and candidate effectors, semiquantitative RT-PCR showed that these transcripts were either expressed constitutively in both in vitro and in planta or induced during plant infection. Using potato virus X (PVX based transient expression assays, we showed that one of the candidate effectors, i. e. contig 8 that encodes a cerato-platanin (CP domain containing protein, unlike CP proteins

  2. Putative thyroid hormone receptors in red blood cells of some reptiles.

    Science.gov (United States)

    Wong, C C; Chiu, K W

    1987-06-01

    Putative triiodothyronine (T3) receptors have been detected in the nuclei of red blood cells (RBC) in a number of reptile species. The binding characteristics of T3 receptors in vitro were dissociation constant (Kd) 9.1 to 28.58, 36.8 and 40, and 11.12 and 11.36 pM, and binding capacity (Bmax) 0.12 to 0.37, 0.17 and 0.24, and 0.19 and 0.28 fmol per million cells in the rat snake (Ptyas korros), soft-shelled turtle (Trionyx sinensis), and tokay gecko (Gekko gecko), respectively. These data were obtained in all species using in vitro incubation of whole cell according to current receptor studies on living cells. With modified technique in subsequent experiments, these values of the binding characteristics were seemingly low. The discrepancy was ascribed to the assessment of "free" fraction of hormone which would be used in subsequent calculation.

  3. Excitatory amino acid b-N-methylamino-L-alanine is a putative environmental neurotoxin

    Directory of Open Access Journals (Sweden)

    VLADIMIR NEDELJKOV

    2011-04-01

    Full Text Available The amino acid b-N-methylamino-L-alanine (L-BMAA has been associated with the amyotrophic lateral sclerosis/parkinsonism-dementia complex in three distinct western Pacific populations. The putative neurotoxin is produced by cyanobacteria, which live symbiotically in the roots of cycad trees. L-BMAA was thought to be a threat only to those few populations whose diet and medicines rely heavily on cycad seeds. However, the recent discovery that cyanobacteria from diverse terrestrial, freshwater, and saltwater ecosystems around the world produce the toxin requires a reassessment of whether it poses a larger health threat. Therefore, it is proposed that monitoring L-BMAA levels in cyanobacteria-contaminated water supplies might be prudent.

  4. Molecular characterization of putative Hepatozoon sp. from the sedge warbler (Acrocephalus schoenobaenus).

    Science.gov (United States)

    Biedrzycka, Aleksandra; Kloch, Agnieszka; Migalska, Magdalena; Bielański, Wojciech

    2013-05-01

    We characterized partial sequences of 18S rDNA from sedge warblers infected with a parasite described previously as Hepatozoon kabeeni. Prevalence was 47% in sampled birds.We detected 3 parasite haplotypes in 62 sequenced samples from infected animals. In phylogenetic analyses, 2 of the putative Hepatozoon haplotypes closely resembled Lankesterella minima and L. valsainensis. The third haplotype grouped in a wider clade composed of Caryospora and Eimeria. None of the haplotypes showed resemblance to sequences of Hepatozoon from reptiles and mammals. Molecular detection results were consistent with those from microscopy of stained blood smears, confirming that the primers indeed amplified the parasite sequences. Here we provide evidence that the avian Hepatozoon-like parasites are most likely Lankesterella, supporting the suggestion that the systematic position of avian Hepatozoon-like species needs to be revised.

  5. A tiered approach to evaluate an iodine recycling inhibition adverse outcome pathway (AOP) in amphibians

    Science.gov (United States)

    The enzyme iodotyrosine deiodinase (dehalogenase, IYD) catalyzes iodide recycling and promotes iodide retention in thyroid follicular cells. Loss of function or chemical inhibition of IYD reduces thyroid hormone synthesis, which leads to insufficiency in tissues and subsequent ne...

  6. Genetic dissimilarity of putative gamma-ray-induced 'Preciosa-AAAB-Pome type' banana (Musa sp) mutants based on multivariate statistical analysis.

    Science.gov (United States)

    Pestana, R K N; Amorim, E P; Ferreira, C F; Amorim, V B O; Oliveira, L S; Ledo, C A S; Silva, S O

    2011-10-25

    Bananas are among the most important fruit crops worldwide, being cultivated in more than 120 countries, mainly by small-scale producers. However, short-stature high-yielding bananas presenting good agronomic characteristics are hard to find. Consequently, wind continues to damage a great number of plantations each year, leading to lodging of plants and bunch loss. Development of new cultivars through conventional genetic breeding methods is hindered by female sterility and the low number of seeds. Mutation induction seems to have great potential for the development of new cultivars. We evaluated genetic dissimilarity among putative 'Preciosa' banana mutants generated by gamma-ray irradiation, using morphoagronomic characteristics and ISSR markers. The genetic distances between the putative 'Preciosa' mutants varied from 0.21 to 0.66, with a cophenetic correlation coefficient of 0.8064. We found good variability after irradiation of 'Preciosa' bananas; this procedure could be useful for banana breeding programs aimed at developing short-stature varieties with good agronomic characteristics.

  7. Transcriptome Analysis of Mango (Mangifera indica L.) Fruit Epidermal Peel to Identify Putative Cuticle-Associated Genes

    Science.gov (United States)

    Tafolla-Arellano, Julio C.; Zheng, Yi; Sun, Honghe; Jiao, Chen; Ruiz-May, Eliel; Hernández-Oñate, Miguel A.; González-León, Alberto; Báez-Sañudo, Reginaldo; Fei, Zhangjun; Domozych, David; Rose, Jocelyn K. C.; Tiznado-Hernández, Martín E.

    2017-04-01

    Mango fruit (Mangifera indica L.) are highly perishable and have a limited shelf life, due to postharvest desiccation and senescence, which limits their global distribution. Recent studies of tomato fruit suggest that these traits are influenced by the expression of genes that are associated with cuticle metabolism. However, studies of these phenomena in mango fruit are limited by the lack of genome-scale data. In order to gain insight into the mango cuticle biogenesis and identify putative cuticle-associated genes, we analyzed the transcriptomes of peels from ripe and overripe mango fruit using RNA-Seq. Approximately 400 million reads were generated and de novo assembled into 107,744 unigenes, with a mean length of 1,717 bp and with this information an online Mango RNA-Seq Database (http://bioinfo.bti.cornell.edu/cgi-bin/mango/index.cgi) which is a valuable genomic resource for molecular research into the biology of mango fruit was created. RNA-Seq analysis suggested that the pathway leading to biosynthesis of the cuticle component, cutin, is up-regulated during overripening. This data was supported by analysis of the expression of several putative cuticle-associated genes and by gravimetric and microscopic studies of cuticle deposition, revealing a complex continuous pattern of cuticle deposition during fruit development and involving substantial accumulation during ripening/overripening.

  8. The putative Leishmania telomerase RNA (LeishTER undergoes trans-splicing and contains a conserved template sequence.

    Directory of Open Access Journals (Sweden)

    Elton J R Vasconcelos

    Full Text Available Telomerase RNAs (TERs are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER that contains a 5' spliced leader (SL cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs and its role in parasite telomere biology.

  9. Formation of putative chloroplast cytochromes in isolated developing pea chloroplasts

    International Nuclear Information System (INIS)

    Thaver, S.S.; Bhava, D.; Castelfranco, P.A.

    1986-01-01

    In addition to chlorophyll-protein complexes, other proteins were labeled when isolated developing pea chloroplasts were incubated with [ 14 C]-5-aminolevulinic acid [ 14 C]-ALA. The major labeled band (M/sub r/ = 43 kDa by LDS-PAGE) was labeled even in the presence of chloramphenicol. Heme-dependent peroxidase activity (as detected by the tetramethyl benzidine-H 2 O 2 stain) was not visibly associated with this band. The radioactive band was stable to heat, 5% HCl in acetone, and was absent if the incubation with [ 14 C]-5-aminolevulinic acid was carried out in the presence of N-methyl protoporphyrin IX dimethyl ester (a specific inhibitor of ferrochelatase). Organic solvent extraction procedures for the enrichment of cytochrome f from chloroplast membranes also extracted this unknown labeled product. It was concluded that this labeled product was probably a c-type cytochrome. The effect of exogenous iron, iron chelators, gabaculine (an inhibitor of ALA synthesis) and other incubation conditions upon the in vitro formation of putative chloroplast cytochromes will be discussed

  10. Primary genome scan to identify putative quantitative trait loci for feedlot growth rate, feed intake, and feed efficiency of beef cattle.

    Science.gov (United States)

    Nkrumah, J D; Sherman, E L; Li, C; Marques, E; Crews, D H; Bartusiak, R; Murdoch, B; Wang, Z; Basarab, J A; Moore, S S

    2007-12-01

    Feed intake and feed efficiency of beef cattle are economically relevant traits. The study was conducted to identify QTL for feed intake and feed efficiency of beef cattle by using genotype information from 100 microsatellite markers and 355 SNP genotyped across 400 progeny of 20 Angus, Charolais, or Alberta Hybrid bulls. Traits analyzed include feedlot ADG, daily DMI, feed-to-gain ratio [F:G, which is the reciprocal of the efficiency of gain (G:F)], and residual feed intake (RFI). A mixed model with sire as random and QTL effects as fixed was used to generate an F-statistic profile across and within families for each trait along each chromosome, followed by empirical permutation tests to determine significance thresholds for QTL detection. Putative QTL for ADG (chromosome-wise P < 0.05) were detected across families on chromosomes 5 (130 cM), 6 (42 cM), 7 (84 cM), 11 (20 cM), 14 (74 cM), 16 (22 cM), 17 (9 cM), 18 (46 cM), 19 (53 cM), and 28 (23 cM). For DMI, putative QTL that exceeded the chromosome-wise P < 0.05 threshold were detected on chromosomes 1 (93 cM), 3 (123 cM), 15 (31 cM), 17 (81 cM), 18 (49 cM), 20 (56 cM), and 26 (69 cM) in the across-family analyses. Putative across-family QTL influencing F:G that exceeded the chromosome-wise P < 0.05 threshold were detected on chromosomes 3 (62 cM), 5 (129 cM), 7 (27 cM), 11 (16 cM), 16 (30 cM), 17 (81 cM), 22 (72 cM), 24 (55 cM), and 28 (24 cM). Putative QTL influencing RFI that exceeded the chromosome-wise P < 0.05 threshold were detected on chromosomes 1 (90 cM), 5 (129 cM), 7 (22 cM), 8 (80 cM), 12 (89 cM), 16 (41 cM), 17 (19 cM), and 26 (48 cM) in the across-family analyses. In addition, a total of 4, 6, 1, and 8 chromosomes showed suggestive evidence (chromosome-wise, P < 0.10) for putative ADG, DMI, F:G, and RFI QTL, respectively. Most of the QTL detected across families were also detected within families, although the locations across families were not necessarily the locations within families, which is

  11. Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma

    International Nuclear Information System (INIS)

    Tong, Joanna H; Lo, Kwok W; To, Ka F; Ng, David C; Chau, Shuk L; So, Ken K; Leung, Patrick P; Lee, Tin L; Lung, Raymond W; Chan, Michael W; Chan, Anthony W

    2010-01-01

    Human Disabled-2 (DAB2), is a multi-function signalling molecule that it is frequently down-regulated in human cancers. We aimed to investigate the possible tumour suppressor effect of DAB2 in nasopharyngeal carcinoma (NPC). We studied the expression of DAB2 in NPC cell lines, xenografts and primary tumour samples. The status of promoter methylation was assessed by methylation specific PCR and bisulfite sequencing. The functional role of DAB2 in NPC was investigated by re-introducing DAB2 expression into NPC cell line C666-1. Decrease or absent of DAB2 transcript was observed in NPC cell lines and xenografts. Loss of DAB2 protein expression was seen in 72% (33/46) of primary NPC as demonstrated by immunohistochemistry. Aberrant DAB2 promoter methylation was detected in 65.2% (30/46) of primary NPC samples by methylation specific PCR. Treatment of the DAB2 negative NPC cell line C666-1 with 5-aza-2'-deoxycytidine resulted in restoration of DAB2 expression in a dose-dependent manner. Overexpression of DAB2 in NPC cell line C666-1 resulted in reduced growth rate and 35% reduction in anchorage-dependent colony formation, and inhibition of serum-induced c-Fos expression compared to vector-transfected controls. Over expression of DAB2 resulted in alterations of multiple pathways as demonstrated by expression profiling and functional network analysis, which confirmed the role of DAB2 as an adaptor molecule involved in multiple receptor-mediated signalling pathways. We report the frequent down regulation of DAB2 in NPC and the promoter hypermethylation contributes to the loss of expression of DAB2. This is the first study demonstrating frequent DAB2 promoter hypermethylation in human cancer. Our functional studies support the putative tumour suppressor effect of DAB2 in NPC cells

  12. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    International Nuclear Information System (INIS)

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group

  13. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    Energy Technology Data Exchange (ETDEWEB)

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Roszak, Aleksander W. [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel, E-mail: daniel.walker@glasgow.ac.uk [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom)

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  14. Structural insights into RipC, a putative citrate lyase β subunit from a Yersinia pestis virulence operon

    International Nuclear Information System (INIS)

    Torres, Rodrigo; Chim, Nicholas; Sankaran, Banumathi; Pujol, Céline; Bliska, James B.; Goulding, Celia W.

    2011-01-01

    Comparison of the 2.45 Å resolution crystal structure of homotrimeric RipC, a putative citrate lyase β subunit from Y. pestis, with structural homologs reveals conserved RipC residues that are implicated in CoA binding. Yersinia pestis remains a threat, with outbreaks of plague occurring in rural areas and its emergence as a weapon of bioterrorism; thus, an improved understanding of its various pathogenicity pathways is warranted. The rip (required for intracellular proliferation) virulence operon is required for Y. pestis survival in interferon-γ-treated macrophages and has been implicated in lowering macrophage-produced nitric oxide levels. RipC, one of three gene products from the rip operon, is annotated as a citrate lyase β subunit. Furthermore, the Y. pestis genome lacks genes that encode citrate lyase α and γ subunits, suggesting a unique functional role of RipC in the Y. pestisrip-mediated survival pathway. Here, the 2.45 Å resolution crystal structure of RipC revealed a homotrimer in which each monomer consists of a (β/α) 8 TIM-barrel fold. Furthermore, the trimeric state was confirmed in solution by size-exclusion chromatography. Through sequence and structure comparisons with homologous proteins, it is proposed that RipC is a putative CoA- or CoA-derivative binding protein

  15. Posttraumatic stress symptoms and tobacco abstinence effects in a non-clinical sample: evaluating the mediating role of negative affect reduction smoking expectancies.

    Science.gov (United States)

    Langdon, Kirsten J; Leventhal, Adam M

    2014-11-01

    The relation between posttraumatic stress symptoms and smoking is well documented but poorly understood. The present investigation sought to evaluate the impact of posttraumatic stress symptoms on subjective and behavioral tobacco abstinence effects both directly and indirectly through negative affect reduction smoking outcome expectancies. Participants included 275 (68.7% male; Mage =43.9, 10+ cig/day) adult non-treatment seeking smokers, who attended two counterbalanced laboratory sessions (16 h of smoking deprivation vs ad libitum smoking), during which they completed self-report measures of withdrawal symptoms and mood followed by a smoking lapse task in which they could earn money for delaying smoking and purchase cigarettes to smoke. Results supported a mediational pathway whereby higher baseline symptoms of posttraumatic stress predicted greater endorsement of expectancies that smoking will effectively reduce negative affect, which in turn predicted greater abstinence-provoked exacerbations in nicotine withdrawal symptoms and negative affect. Posttraumatic stress symptoms also predicted number of cigarettes purchased independent of negative affect reduction expectancies, but did not predict delaying smoking for money. Findings highlight tobacco abstinence effects as a putative mechanism underlying posttraumatic stress disorder (PTSD)-smoking comorbidity, indicate an important mediating role of beliefs for smoking-induced negative affect reduction, and shed light on integrated treatment approaches for these two conditions. © The Author(s) 2014.

  16. A G-protein-coupled chemokine receptor: A putative insertion site for a multi-pathogen recombinant capripoxvirus vaccine strategy.

    Science.gov (United States)

    Cêtre-Sossah, Catherine; Dickmu, Simon; Kwiatek, Olivier; Albina, Emmanuel

    2017-09-01

    Capripoxviruses (CaPVs) have been shown to be ideal viral vectors for the development of recombinant multivalent vaccines to enable delivery of immunogenic genes from ruminant pathogens. So far, the viral thymidine kinase (TK) gene is the only gene used to generate recombinants. A putative non-essential gene encoding a G-protein-coupled chemokine receptor subfamily homologue (GPCR) was targeted as an additional insertion site. Peste des petits ruminants (PPR) was chosen as a disease model. A new recombinant CaPV expressing the viral attachment hemagglutinin (H) of the PPR virus (PPRV) in the GPCR insertion site (rKS1-HPPR-GPCR) was generated in the backbone North African isolate KS1 strain of lumpy skin disease virus (LSDV). Comparison with the recombinant CaPV expressing the H of PPRV in the TK gene (rKS1-HPPR-TK) shown to induce protection against both PPR and LSD in both sheep and goats was assessed. The suitability of the GPCR gene to be a putative additional insertion site in the CaPV genome is evaluated and discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. An acyltransferase gene that putatively functions in anthocyanin modification was horizontally transferred from Fabaceae into the genus Cuscuta

    Directory of Open Access Journals (Sweden)

    Ting Sun

    2016-06-01

    Full Text Available Horizontal gene transfer (HGT refers to the flow of genetic materials to non-offspring, and occasionally HGT in plants can improve the adaptation of organisms in new niches due to expanded metabolic capability. Anthocyanins are an important group of water-soluble red, purple, or blue secondary metabolites, whose diversity results from modification after the main skeleton biosynthesis. Cuscuta is a stem holoparasitic genus, whose members form direct connection with hosts to withdraw water, nutrients, and macromolecules. Such intimate association is thought to increase the frequency of HGT. By transcriptome screening for foreign genes in Cuscuta australis, we discovered that one gene encoding a putative anthocyanin acyltransferase gene of the BAHD family, which is likely to be involved in anthocyanin modification, was acquired by C. australis from Fabaceae through HGT. The anthocyanin acyltransferase-like (AT-like gene was confirmed to be present in the genome assembly of C. australis and the transcriptomes of Cuscuta pentagona. The higher transcriptional level in old stems is consistent with its putative function in secondary metabolism by stabilizing anthocyanin at neutral pH and thus HGT of this AT-like gene may have improved biotic and abiotic resistance of Cuscuta.

  18. RPS6KA2, a putative tumour suppressor gene at 6q27 in sporadic epithelial ovarian cancer

    DEFF Research Database (Denmark)

    Bignone, P A; Lee, K Y; Liu, Y

    2007-01-01

    We had previously defined by allele loss studies a minimal region at 6q27 (between D6S264 and D6S297) to contain a putative tumour suppressor gene. The p90 ribosomal S6 kinase-3 gene (p90 Rsk-3, RPS6KA2) maps in this interval. It is a serine-threonine kinase that signals downstream of the mitogen...

  19. The Effect of Carbonaceous Reductant Selection on Chromite Pre-reduction

    Science.gov (United States)

    Kleynhans, E. L. J.; Beukes, J. P.; Van Zyl, P. G.; Bunt, J. R.; Nkosi, N. S. B.; Venter, M.

    2017-04-01

    Ferrochrome (FeCr) production is an energy-intensive process. Currently, the pelletized chromite pre-reduction process, also referred to as solid-state reduction of chromite, is most likely the FeCr production process with the lowest specific electricity consumption, i.e., MWh/t FeCr produced. In this study, the effects of carbonaceous reductant selection on chromite pre-reduction and cured pellet strength were investigated. Multiple linear regression analysis was employed to evaluate the effect of reductant characteristics on the aforementioned two parameters. This yielded mathematical solutions that can be used by FeCr producers to select reductants more optimally in future. Additionally, the results indicated that hydrogen (H)- (24 pct) and volatile content (45.8 pct) were the most significant contributors for predicting variance in pre-reduction and compressive strength, respectively. The role of H within this context is postulated to be linked to the ability of a reductant to release H that can induce reduction. Therefore, contrary to the current operational selection criteria, the authors believe that thermally untreated reductants ( e.g., anthracite, as opposed to coke or char), with volatile contents close to the currently applied specification (to ensure pellet strength), would be optimal, since it would maximize H content that would enhance pre-reduction.

  20. The Study of NADPH-Dependent Flavoenzyme-Catalyzed Reduction of Benzo[1,2-c]1,2,5-oxadiazole N-Oxides (Benzofuroxans

    Directory of Open Access Journals (Sweden)

    Jonas Šarlauskas

    2014-12-01

    Full Text Available The enzymatic reactivity of a series of benzo[1,2-c]1,2,5-oxadiazole N-oxides (benzofuroxans; BFXs towards mammalian single-electron transferring NADPH:cytochrome P-450 reductase (P-450R and two-electron (hydride transferring NAD(PH:quinone oxidoreductase (NQO1 was examined in this work. Since the =N+ (→OO− moiety of furoxan fragments of BFXs bears some similarity to the aromatic nitro-group, the reactivity of BFXs was compared to that of nitro-aromatic compounds (NACs whose reduction mechanisms by these and other related flavoenzymes have been extensively investigated. The reduction of BFXs by both P-450R and NQO1 was accompanied by O2 uptake, which was much lower than the NADPH oxidation rate; except for annelated BFXs, whose reduction was followed by the production of peroxide. In order to analyze the possible quantitative structure-activity relationships (QSARs of the enzymatic reactivity of the compounds, their electron-accepting potency and other reactivity indices were assessed by quantum mechanical methods. In P-450R-catalyzed reactions, both BFXs and NACs showed the same reactivity dependence on their electron-accepting potency which might be consistent with an “outer sphere” electron transfer mechanism. In NQO1-catalyzed two-electron (hydride transferring reactions, BFXs acted as more efficient substrates than NACs, and the reduction efficacy of BFXs by NQO1 was in general higher than by single-electron transferring P-450R. In NQO1-catalyzed reactions, QSARs obtained showed that the reduction efficacy of BFXs, as well as that of NACs, was determined by their electron-accepting potency and could be influenced by their binding mode in the active center of NQO1 and by their global softness as their electronic characteristic. The reductive conversion of benzofuroxan by both flavoenzymes yielded the same reduction product of benzofuroxan, 2,3-diaminophenazine, with the formation of o-benzoquinone dioxime as a putative primary

  1. Cloning of 1,2-Dichloroethane Degradation Genes of Xanthobacter autotrophicus GJ10 and Expression and Sequencing of the dhlA Gene

    NARCIS (Netherlands)

    Janssen, Dick B.; Pries, Frens; Ploeg, Jan van der; Kazemier, Bert; Terpstra, Peter; Witholt, Bernard

    1989-01-01

    A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in

  2. Ligand binding analyses of the putative peptide transporter YjdL from E. coli display a significant selectivity towards dipeptides

    DEFF Research Database (Denmark)

    Ernst, Heidi Asschenfeldt; Pham, Antony; Hald, Helle

    2009-01-01

    Proton-dependent oligopeptide transporters (POTs) are secondary active transporters that couple the inwards translocation of di- and tripeptides to inwards proton translocation. Escherichia coli contains four genes encoding the putative POT proteins YhiP, YdgR, YjdL and YbgH. We have over-express...

  3. Identification and characterization of a putative human platelet thromboxane A2/prostaglandin H2 receptor

    International Nuclear Information System (INIS)

    Saussy, D.L. Jr.

    1986-01-01

    The thromboxane A 2 (TXA 2 ) analog, 9,11-dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13,14-dihydro-13-aza-15αβ-omega-tetranor TXA 2 (I-PTA-OH) was characterized as a competitive antagonist of TXA 2 mimetic-induced platelet aggregation, with a K/sub d/ of 190 nM in platelet rich plasma. This antagonism was specific for the putative thromboxane A 2 /prostaglandin H 2 (TXA 2 /PGH 2 ) receptor, since I-PTA-OH had no inhibitory effects on platelet aggregation stimulated by agonists which act independently of TXA 2 /PGH 2 , and did not inhibit platelet TXA 2 synthesis. [ 125 I]-PTA-OH binding to a particulate fraction from human platelets was saturable, displaceable, and linear with protein concentration. Scatchard analysis of equilibrium binding revealed a single class of high affinity binding sites, with a K/sub d/ of 30 +/- 4 nM and a B/sub max/ of 1.8 +/- 0.3 pmol/mg protein. Kinetic analysis yielded a k 1 of 1.35 x 10 6 M -1 x min -1 and a k√ 1 of 0.032 min -1 , K/sub d/ = k√ 1 /k 1 = 24 nM. The subcellular localization of the putative TXA 2 /PGH 2 receptor was determined using [ 125 I]-PTA-OH binding as a marker for the receptor. [ 125 I]-PTA-OH binding as a marker for the receptor. [ 125 I]-PTA-OH binding, was coenriched with markers for plasma membranes and dense tubular system; but not with markers for cytoplasmic constituents, mitochondria, or granules

  4. Genomic characterization of putative allergen genes in peach/almond and their synteny with apple

    Science.gov (United States)

    Chen, Lin; Zhang, Shuiming; Illa, Eudald; Song, Lijuan; Wu, Shandong; Howad, Werner; Arús, Pere; Weg, Eric van de; Chen, Kunsong; Gao, Zhongshan

    2008-01-01

    Background Fruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4) have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica), these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcis)Pru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica). Results Eight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1). Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B) were on G3, Pru p/du 2.02 on G7,Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01–3.03) containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02) were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2. Conclusion A total of 18 putative peach/almond allergen genes have

  5. Genomic characterization of putative allergen genes in peach/almond and their synteny with apple

    Directory of Open Access Journals (Sweden)

    Weg Eric

    2008-11-01

    Full Text Available Abstract Background Fruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4 have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica, these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcisPru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica. Results Eight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1. Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B were on G3, Pru p/du 2.02 on G7,Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01–3.03 containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02 were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2. Conclusion A total of 18 putative peach

  6. SACE_5599, a putative regulatory protein, is involved in morphological differentiation and erythromycin production in Saccharopolyspora erythraea.

    Science.gov (United States)

    Kirm, Benjamin; Magdevska, Vasilka; Tome, Miha; Horvat, Marinka; Karničar, Katarina; Petek, Marko; Vidmar, Robert; Baebler, Spela; Jamnik, Polona; Fujs, Štefan; Horvat, Jaka; Fonovič, Marko; Turk, Boris; Gruden, Kristina; Petković, Hrvoje; Kosec, Gregor

    2013-12-17

    Erythromycin is a medically important antibiotic, biosynthesized by the actinomycete Saccharopolyspora erythraea. Genes encoding erythromycin biosynthesis are organized in a gene cluster, spanning over 60 kbp of DNA. Most often, gene clusters encoding biosynthesis of secondary metabolites contain regulatory genes. In contrast, the erythromycin gene cluster does not contain regulatory genes and regulation of its biosynthesis has therefore remained poorly understood, which has for a long time limited genetic engineering approaches for erythromycin yield improvement. We used a comparative proteomic approach to screen for potential regulatory proteins involved in erythromycin biosynthesis. We have identified a putative regulatory protein SACE_5599 which shows significantly higher levels of expression in an erythromycin high-producing strain, compared to the wild type S. erythraea strain. SACE_5599 is a member of an uncharacterized family of putative regulatory genes, located in several actinomycete biosynthetic gene clusters. Importantly, increased expression of SACE_5599 was observed in the complex fermentation medium and at controlled bioprocess conditions, simulating a high-yield industrial fermentation process in the bioreactor. Inactivation of SACE_5599 in the high-producing strain significantly reduced erythromycin yield, in addition to drastically decreasing sporulation intensity of the SACE_5599-inactivated strains when cultivated on ABSM4 agar medium. In contrast, constitutive overexpression of SACE_5599 in the wild type NRRL23338 strain resulted in an increase of erythromycin yield by 32%. Similar yield increase was also observed when we overexpressed the bldD gene, a previously identified regulator of erythromycin biosynthesis, thereby for the first time revealing its potential for improving erythromycin biosynthesis. SACE_5599 is the second putative regulatory gene to be identified in S. erythraea which has positive influence on erythromycin yield. Like bld

  7. Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics

    DEFF Research Database (Denmark)

    Gravesen, Anne; Sorensen, K.; Aarestrup, Frank Møller

    2001-01-01

    -molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology), The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wildtype strains, indicating...

  8. Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

    Science.gov (United States)

    Li, Yuanjun; Gou, Junbo; Chen, Fangfang; Li, Changfu; Zhang, Yansheng

    2016-01-01

    Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones (STLs), which include the xanthanolides. To date, the biogenesis of xanthanolides, especially their downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that are highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of STLs are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides.

  9. Sensitivity analysis of the relationship between disease occurrence and distance from a putative source of pollution

    Directory of Open Access Journals (Sweden)

    Emanuela Dreassi

    2008-05-01

    Full Text Available The relation between disease risk and a point source of pollution is usually investigated using distance from the source as a proxy of exposure. The analysis may be based on case-control data or on aggregated data. The definition of the function relating risk of disease and distance is critical, both in a classical and in a Bayesian framework, because the likelihood is usually very flat, even with large amounts of data. In this paper we investigate how the specification of the function relating risk of disease with distance from the source and of the prior distributions on the parameters of the function affects the results when case-control data and Bayesian methods are used. We consider different popular parametric models for the risk distance function in a Bayesian approach, comparing estimates with those derived by maximum likelihood. As an example we have analyzed the relationship between a putative source of environmental pollution (an asbestos cement plant and the occurrence of pleural malignant mesothelioma in the area of Casale Monferrato (Italy in 1987-1993. Risk of pleural malignant mesothelioma turns out to be strongly related to distance from the asbestos cement plant. However, as the models appeared to be sensitive to modeling choices, we suggest that any analysis of disease risk around a putative source should be integrated with a careful sensitivity analysis and possibly with prior knowledge. The choice of prior distribution is extremely important and should be based on epidemiological considerations.

  10. Aspergillus nidulans Natural Product Biosynthesis Is Regulated by MpkB, a Putative Pheromone Response Mitogen-Activated Protein Kinase

    International Nuclear Information System (INIS)

    Atoui, A.; Bao, D.; Kaur, N.; Grayburn, W.S.; Calvo, A.M.

    2008-01-01

    The Aspergillus nidulans putative mitogen-activated protein kinase encoded by mpkB has a role in natural product biosynthesis. An mpkB mutant exhibited a decrease in sterigmatocystin gene expression and low mycotoxin levels. The mutation also affected the expression of genes involved in penicillin and terrequinone A synthesis. mpkB was necessary for normal expression of laeA, which has been found to regulate secondary metabolism gene clusters. (author)

  11. Expression of putative immune response genes during early ontogeny in the coral Acropora millepora.

    Directory of Open Access Journals (Sweden)

    Eneour Puill-Stephan

    Full Text Available Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals.Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR. Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A. millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria during winnowing processes as symbioses are fine-tuned.Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are

  12. Expression of putative immune response genes during early ontogeny in the coral Acropora millepora.

    Science.gov (United States)

    Puill-Stephan, Eneour; Seneca, François O; Miller, David J; van Oppen, Madeleine J H; Willis, Bette L

    2012-01-01

    Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals. Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A. millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned. Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further

  13. Shewanella putrefaciens mtrB encodes an outer membrane protein required for Fe(III) and Mn(IV) reduction.

    Science.gov (United States)

    Beliaev, A S; Saffarini, D A

    1998-12-01

    Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.

  14. Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-l-homocysteine

    International Nuclear Information System (INIS)

    Pioszak, Augen A.; Murayama, Kazutaka; Nakagawa, Noriko; Ebihara, Akio; Kuramitsu, Seiki; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2005-01-01

    Three structures of a putative RNA 5-methyluridine methyltransferase from T. thermophilus, including its complex with S-adenosyl-l-homocysteine, are presented. The structures reveal the mode of cofactor binding, architecture of the putative active site, and the presence of a deep cleft adjacent to the active site that may bind RNA. The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-l-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-l-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 Å resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 Å, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central α/β-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.

  15. Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways

    NARCIS (Netherlands)

    Poelarends, GJ; Kulakov, LA; Larkin, MJ; van Hylckama Vlieg, Johan E.T.; Janssen, DB

    The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct

  16. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Chemical Sciences. Dhurairajan senthilnathan. Articles written in Journal of Chemical Sciences. Volume 123 Issue 3 May 2011 pp 279-290. Biocatalysis of azidolysis of epoxides: Computational evidences on the role of halohydrin dehalogenase (HheC) · Dhurairajan senthilnathan ...

  17. Expression, purification, crystallization and preliminary X-ray characterization of a putative glycosyltransferase of the GT-A fold found in mycobacteria

    Energy Technology Data Exchange (ETDEWEB)

    Fulton, Zara [The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia); Crellin, Paul K.; Brammananth, Rajini [Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia); Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Zaker-Tabrizi, Leyla [The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia); Coppel, Ross L. [Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia); Department of Microbiology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Rossjohn, Jamie, E-mail: jamie.rossjohn@med.monash.edu.au; Beddoe, Travis, E-mail: jamie.rossjohn@med.monash.edu.au [The Protein Crystallography Unit, Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800 (Australia); Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, Victoria 3800 (Australia)

    2008-05-01

    MAP2569c from M. avium subsp. paratuberculosis, a putative glycosyltransferase implicated in mycobacterial cell-wall biosynthesis, was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution. Glycosidic bond formation is a ubiquitous enzyme-catalysed reaction. This glycosyltransferase-mediated process is responsible for the biosynthesis of innumerable oligosaccharides and glycoconjugates and is often organism- or cell-specific. However, despite the abundance of genomic information on glycosyltransferases (GTs), there is a lack of structural data for this versatile class of enzymes. Here, the cloning, expression, purification and crystallization of an essential 329-amino-acid (34.8 kDa) putative GT of the classic GT-A fold implicated in mycobacterial cell-wall biosynthesis are reported. Crystals of MAP2569c from Mycobacterium avium subsp. paratuberculosis were grown in 1.6 M monoammonium dihydrogen phosphate and 0.1 M sodium citrate pH 5.5. A complete data set was collected to 1.8 Å resolution using synchrotron radiation from a crystal belonging to space group P4{sub 1}2{sub 1}2.

  18. Multilocus sequence data reveal dozens of putative cryptic species in a radiation of endemic Californian mygalomorph spiders (Araneae, Mygalomorphae, Nemesiidae).

    Science.gov (United States)

    Leavitt, Dean H; Starrett, James; Westphal, Michael F; Hedin, Marshal

    2015-10-01

    We use mitochondrial and multi-locus nuclear DNA sequence data to infer both species boundaries and species relationships within California nemesiid spiders. Higher-level phylogenetic data show that the California radiation is monophyletic and distantly related to European members of the genus Brachythele. As such, we consider all California nemesiid taxa to belong to the genus Calisoga Chamberlin, 1937. Rather than find support for one or two taxa as previously hypothesized, genetic data reveal Calisoga to be a species-rich radiation of spiders, including perhaps dozens of species. This conclusion is supported by multiple mitochondrial barcoding analyses, and also independent analyses of nuclear data that reveal general genealogical congruence. We discovered three instances of sympatry, and genetic data indicate reproductive isolation when in sympatry. An examination of female reproductive morphology does not reveal species-specific characters, and observed male morphological differences for a subset of putative species are subtle. Our coalescent species tree analysis of putative species lays the groundwork for future research on the taxonomy and biogeographic history of this remarkable endemic radiation. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Expression, purification, crystallization and preliminary X-ray characterization of a putative glycosyltransferase of the GT-A fold found in mycobacteria

    International Nuclear Information System (INIS)

    Fulton, Zara; Crellin, Paul K.; Brammananth, Rajini; Zaker-Tabrizi, Leyla; Coppel, Ross L.; Rossjohn, Jamie; Beddoe, Travis

    2008-01-01

    MAP2569c from M. avium subsp. paratuberculosis, a putative glycosyltransferase implicated in mycobacterial cell-wall biosynthesis, was cloned, expressed, purified and crystallized. X-ray diffraction data were collected to 1.8 Å resolution. Glycosidic bond formation is a ubiquitous enzyme-catalysed reaction. This glycosyltransferase-mediated process is responsible for the biosynthesis of innumerable oligosaccharides and glycoconjugates and is often organism- or cell-specific. However, despite the abundance of genomic information on glycosyltransferases (GTs), there is a lack of structural data for this versatile class of enzymes. Here, the cloning, expression, purification and crystallization of an essential 329-amino-acid (34.8 kDa) putative GT of the classic GT-A fold implicated in mycobacterial cell-wall biosynthesis are reported. Crystals of MAP2569c from Mycobacterium avium subsp. paratuberculosis were grown in 1.6 M monoammonium dihydrogen phosphate and 0.1 M sodium citrate pH 5.5. A complete data set was collected to 1.8 Å resolution using synchrotron radiation from a crystal belonging to space group P4 1 2 1 2

  20. A putative Lynch syndrome family carrying MSH2 and MSH6 variants of uncertain significance-functional analysis reveals the pathogenic one

    DEFF Research Database (Denmark)

    Kantelinen, Jukka; Hansen, Thomas V O; Kansikas, Minttu

    2011-01-01

    Inherited pathogenic mutations in the mismatch repair (MMR) genes, MSH2, MLH1, MSH6, and PMS2 predispose to Lynch syndrome (LS). However, the finding of a variant or variants of uncertain significance (VUS) in affected family members complicates the risk assessment. Here, we describe a putative LS...

  1. The putative E3 ubiquitin ligase ECERIFERUM9 regulates abscisic acid biosynthesis and response during seed germination and postgermination growth in arabidopsis

    KAUST Repository

    Zhao, Huayan; Zhang, Huoming; Cui, Peng; Ding, Feng; Wang, Guangchao; Li, Rongjun; Jenks, Matthew A.; Lü , Shiyou; Xiong, Liming

    2014-01-01

    The ECERIFERUM9 (CER9) gene encodes a putative E3 ubiquitin ligase that functions in cuticle biosynthesis and the maintenance of plant water status. Here, we found that CER9 is also involved in abscisic acid (ABA) signaling in seeds and young

  2. The impact of bioaugmentation on dechlorination kinetics and on microbial dechlorinating communities in subsurface clay till

    International Nuclear Information System (INIS)

    Bælum, Jacob; Scheutz, Charlotte; Chambon, Julie C.; Jensen, Christine Mosegaard; Brochmann, Rikke P.; Dennis, Philip; Laier, Troels; Broholm, Mette M.; Bjerg, Poul L.; Binning, Philip J.; Jacobsen, Carsten S.

    2014-01-01

    A molecular study on how the abundance of the dechlorinating culture KB-1 affects dechlorination rates in clay till is presented. DNA extracts showed changes in abundance of specific dechlorinators as well as their functional genes. Independently of the KB-1 added, the microbial dechlorinator abundance increased to the same level in all treatments. In the non-bioaugmented microcosms the reductive dehalogenase gene bvcA increased in abundance, but when KB-1 was added the related vcrA gene increased while bvcA genes did not increase. Modeling showed higher vinyl-chloride dechlorination rates and shorter time for complete dechlorination to ethene with higher initial concentration of KB-1 culture, while cis-dichloroethene dechlorination rates were not affected by KB-1 concentrations. This study provides high resolution abundance profiles of Dehalococcoides spp. (DHC) and functional genes, highlights the ecological behavior of KB-1 in clay till, and reinforces the importance of using multiple functional genes as biomarkers for reductive dechlorination. -- Highlights: • vcrA gene is not always linked to reductive dechlorination potential. • High concentrations of KB-1 stimulate vinyl-chloride degradation. • Vinyl-chloride degradation in non-bioaugmented aquifer is linked to bvcA gene. -- vcrA gene biomarker for reductive dechlorination must be supplemented by bvcA and KB-1 had a positive effect on vinyl-chloride dechlorination compared to dichloroethene dechlorination

  3. Natural Leishmania (Viannia) spp. infections in phlebotomine sand flies (Diptera: Psychodidae) from the Brazilian Amazon region reveal new putative transmission cycles of American cutaneous leishmaniasis

    Science.gov (United States)

    de Souza, Adelson Alcimar Almeida; dos Santos, Thiago Vasconcelos; Jennings, Yara Lúcia Lins; Ishikawa, Edna Aoba Yassui; Barata, Iorlando da Rocha; Silva, Maria das Graças Soares; Lima, José Aprígio Nunes; Shaw, Jeffrey; Lainson, Ralph; Silveira, Fernando Tobias

    2016-01-01

    In Amazonian Brazil the etiological agents of American cutaneous leishmaniasis (ACL) belong to at least seven Leishmania species but little is known about the putative phlebotomine sand fly vectors in different biomes. In 2002–2003 a survey of the phlebotomine fauna was undertaken in the “Floresta Nacional do Tapajós”, Belterra municipality, in the lower Amazon region, western Pará State, Brazil, where we recently confirmed the presence of a putative hybrid parasite, L. (V.) guyanensis × L. (V.) shawi shawi. Sand flies were collected from Centers for Disease Control (CDC) light traps, Shannon traps and by aspiration on tree bases. Females were dissected and attempts to isolate any flagellate infections were made by inoculating homogenized midguts into Difco B45 medium. Isolates were characterized by monoclonal antibodies and isoenzyme electrophoresis. A total of 9,704 sand flies, belonging to 68 species or subspecies, were collected. Infections were found in the following sand flies: L. (V.) naiffi with Psychodopygus hirsutus hirsutus (1) and Ps. davisi (2); and L. (V.) shawi shawi with Nyssomyia whitmani (3) and Lutzomyia gomezi (1). These results provide strong evidence of new putative transmission cycles for L. (V.) naiffi and L. (V.) s. shawi. PMID:27235194

  4. The two putative comS homologs of the biotechnologically important Bacillus licheniformis do not contribute to competence development.

    Science.gov (United States)

    Jakobs, Mareike; Hoffmann, Kerstin; Liesegang, Heiko; Volland, Sonja; Meinhardt, Friedhelm

    2015-03-01

    In Bacillus subtilis, natural genetic competence is subject to complex genetic regulation and quorum sensing dependent. Upon extracellular accumulation of the peptide-pheromone ComX, the membrane-bound sensor histidine kinase ComP initiates diverse signaling pathways by activating-among others-DegQ and ComS. While DegQ favors the expression of extracellular enzymes rather than competence development, ComS is crucial for competence development as it prevents proteolytic degradation of ComK, the key transcriptional activator of all genes required for the uptake and integration of DNA. In Bacillus licheniformis, ComX/ComP sensed cell density negatively influences competence development, suggesting differences from the quorum-sensing-dependent control mechanism in Bacillus subtilis. Here, we show that each of six investigated strains possesses both of two different, recently identified putative comS genes. When expressed from an inducible promoter, none of the comS candidate genes displayed an impact on competence development neither in B. subtilis nor in B. licheniformis. Moreover, disruption of the genes did not reduce transformation efficiency. While the putative comS homologs do not contribute to competence development, we provide evidence that the degQ gene as for B. subtilis negatively influences genetic competency in B. licheniformis.

  5. Distribution of Mast Cells and Locations, Depths, and Sizes of the Putative Acupoints CV 8 and KI 16

    Directory of Open Access Journals (Sweden)

    Sharon Jiyoon Jung

    2017-01-01

    Full Text Available The anatomical locations and sizes of acupuncture points (APs are identified in traditional Chinese medicine by using the cun measurement method. More precise knowledge of those locations and sizes to submillimeter precision, along with their cytological characterizations, would provide significant contributions both to scientific investigations and to precise control of the practice of acupuncture. Over recent decades, researchers have come to realize that APs in the skin of rats and humans have more mast cells (MCs than neighboring nonacupoints. In this work, the distribution of MCs in the ventral skin of mice was studied so that it could be used to infer the locations, depths from the epidermis, and sizes of three putative APs. The umbilicus was taken as the reference point, and a transversal cross section through it was studied. The harvested skins from 8-week-old mice were stained with toluidine blue, and the MCs were recognized by their red-purple stains and their metachromatic granules. The three putative APs, CV 8 and the left and the right KI 16 APs, were identified based on their high densities of MCs. These findings also imply that acupuncture may stimulate, through MCs, an immune response to allergic inflammation.

  6. Infinitary Combinatory Reduction Systems: Normalising Reduction Strategies

    NARCIS (Netherlands)

    Ketema, J.; Simonsen, Jakob Grue

    2010-01-01

    We study normalising reduction strategies for infinitary Combinatory Reduction Systems (iCRSs). We prove that all fair, outermost-fair, and needed-fair strategies are normalising for orthogonal, fully-extended iCRSs. These facts properly generalise a number of results on normalising strategies in

  7. Transcriptomic analysis on the formation of the viable putative non-culturable state of beer-spoilage Lactobacillus acetotolerans

    OpenAIRE

    Junyan Liu; Yang Deng; Brian M. Peters; Lin Li; Bing Li; Lequn Chen; Zhenbo Xu; Mark E. Shirtliff

    2016-01-01

    Lactic acid bacteria (LAB) are the most common beer-spoilage bacteria regardless of beer type, and thus pose significant problems for the brewery industry. The aim of this study was to investigate the genetic mechanisms involved in the ability of the hard-to-culture beer-spoilage bacterium Lactobacillus acetotolerans to enter into the viable putative non-culturable (VPNC) state. A genome-wide transcriptional analysis of beer-spoilage L. acetotolerans strains BM-LA14526, BM-LA14527, and BM-LA1...

  8. Comparative Analysis of Putative Orthologues of Mitochondrial Import Motor Subunit: Pam18 and Pam16 in Plants

    OpenAIRE

    Chen, Xuejin; Ghazanfar, Bushra; Khan, Abdul Rehman; Hayat, Sikandar; Cheng, Zhihui

    2013-01-01

    Pam18/Tim14 and Pam16/Tim16, highly conserved proteins among eukaryotes, are two essential subunits of protein import motors localized in the inner mitochondrial membrane. The heterodimer formed by Pam18 and Pam16 via their J-type domains serves a regulatory function in protein translocation. Here, we report that thirty-one Pam18 and twenty-six Pam16 putative orthologues in twelve plant species were identified and analyzed through bioinformatics strategy. Results data revealed that Pam18 and ...

  9. Polymorphism screening of four genes encoding advanced glycation end-product putative receptors. Association study with nephropathy in type 1 diabetic patients

    DEFF Research Database (Denmark)

    Poirier, Odette; Nicaud, Viviane; Vionnet, N

    2001-01-01

    Advanced glycation end-products (AGEs) may play an important role in the pathogenesis and progression of cardiovascular and renal complications of diabetes. Four putative AGE receptors (RAGEs), AGE-R1, AGE-R2, and AGE-R3 have been described. In this study, we scanned the sequence of the genes enc...

  10. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the soluble domain of PPA0092, a putative nitrite reductase from Propionibacterium acnes

    International Nuclear Information System (INIS)

    Nojiri, Masaki; Shirota, Felicia; Hira, Daisuke; Suzuki, Shinnichiro

    2009-01-01

    The soluble domain of a putative copper-containing nitrite reductase from P. acnes has been overexpressed, purified and crystallized. The crystal belonged to space group P2 1 3 and diffracted to 2.4 Å resolution. The soluble domain (residues 483–913) of PPA0092, a putative copper-containing nitrite reductase from Propionibacterium acnes KPA171202, has been overexpressed in Escherichia coli. The purified recombinant protein was crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected and processed to a maximum resolution of 2.4 Å. The crystal belonged to space group P2 1 3, with unit-cell parameters a = b = c = 108.63 Å. Preliminary diffraction data show that one molecule is present in the asymmetric unit; this corresponds to a V M of 2.1 Å 3 Da −1

  11. High amino acid diversity and positive selection at a putative coral immunity gene (tachylectin-2

    Directory of Open Access Journals (Sweden)

    Hellberg Michael E

    2010-05-01

    Full Text Available Abstract Background Genes involved in immune functions, including pathogen recognition and the activation of innate defense pathways, are among the most genetically variable known, and the proteins that they encode are often characterized by high rates of amino acid substitutions, a hallmark of positive selection. The high levels of variation characteristic of immunity genes make them useful tools for conservation genetics. To date, highly variable immunity genes have yet to be found in corals, keystone organisms of the world's most diverse marine ecosystem, the coral reef. Here, we examine variation in and selection on a putative innate immunity gene from Oculina, a coral genus previously used as a model for studies of coral disease and bleaching. Results In a survey of 244 Oculina alleles, we find high nonsynonymous variation and a signature of positive selection, consistent with a putative role in immunity. Using computational protein structure prediction, we generate a structural model of the Oculina protein that closely matches the known structure of tachylectin-2 from the Japanese horseshoe crab (Tachypleus tridentatus, a protein with demonstrated function in microbial recognition and agglutination. We also demonstrate that at least three other genera of anthozoan cnidarians (Acropora, Montastrea and Nematostella possess proteins structurally similar to tachylectin-2. Conclusions Taken together, the evidence of high amino acid diversity, positive selection and structural correspondence to the horseshoe crab tachylectin-2 suggests that this protein is 1 part of Oculina's innate immunity repertoire, and 2 evolving adaptively, possibly under selective pressure from coral-associated microorganisms. Tachylectin-2 may serve as a candidate locus to screen coral populations for their capacity to respond adaptively to future environmental change.

  12. Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata

    Directory of Open Access Journals (Sweden)

    Tajammul Hussain

    2018-05-01

    Full Text Available The liverwort Radula marginata belongs to the bryophyte division of land plants and is a prospective alternate source of cannabinoid-like compounds. However, mechanistic insights into the molecular pathways directing the synthesis of these cannabinoid-like compounds have been hindered due to the lack of genetic information. This prompted us to do deep sequencing, de novo assembly and annotation of R. marginata transcriptome, which resulted in the identification and validation of the genes for cannabinoid biosynthetic pathway. In total, we have identified 11,421 putative genes encoding 1,554 enzymes from 145 biosynthetic pathways. Interestingly, we have identified all the upstream genes of the central precursor of cannabinoid biosynthesis, cannabigerolic acid (CBGA, including its two first intermediates, stilbene acid (SA and geranyl diphosphate (GPP. Expression of all these genes was validated using quantitative real-time PCR. We have characterized the protein structure of stilbene synthase (STS, which is considered as a homolog of olivetolic acid in R. marginata. Moreover, the metabolomics approach enabled us to identify CBGA-analogous compounds using electrospray ionization mass spectrometry (ESI-MS/MS and gas chromatography mass spectrometry (GC-MS. Transcriptomic analysis revealed 1085 transcription factors (TF from 39 families. Comparative analysis showed that six TF families have been uniquely predicted in R. marginata. In addition, the bioinformatics analysis predicted a large number of simple sequence repeats (SSRs and non-coding RNAs (ncRNAs. Our results collectively provide mechanistic insights into the putative precursor genes for the biosynthesis of cannabinoid-like compounds and a novel transcriptomic resource for R. marginata. The large-scale transcriptomic resource generated in this study would further serve as a reference transcriptome to explore the Radulaceae family.

  13. Biomarkers’ Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM

    Directory of Open Access Journals (Sweden)

    Gretchen L. W. Heavner

    2018-02-01

    Full Text Available Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1TM, including several reductive dehalogenases (RDases and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR as a targeted approach for quantifying transcript copies in the KB-1TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10−12 to 5.9 × 10−10 microelectron equivalents per cell per hour (μeeq/cell∙h. Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA and the hydrogenase HupL (R2 = 0.83 and 0.88, respectively, but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1TM. Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1TM. The addition of oxygen induced cell stress that caused respiration

  14. Biomarkers' Responses to Reductive Dechlorination Rates and Oxygen Stress in Bioaugmentation Culture KB-1TM.

    Science.gov (United States)

    Heavner, Gretchen L W; Mansfeldt, Cresten B; Debs, Garrett E; Hellerstedt, Sage T; Rowe, Annette R; Richardson, Ruth E

    2018-02-08

    Using mRNA transcript levels for key functional enzymes as proxies for the organohalide respiration (OHR) rate, is a promising approach for monitoring bioremediation populations in situ at chlorinated solvent-contaminated field sites. However, to date, no correlations have been empirically derived for chlorinated solvent respiring, Dehalococcoides mccartyi (DMC) containing, bioaugmentation cultures. In the current study, genome-wide transcriptome and proteome data were first used to confirm the most highly expressed OHR-related enzymes in the bioaugmentation culture, KB-1 TM , including several reductive dehalogenases (RDases) and a Ni-Fe hydrogenase, Hup. Different KB-1™ DMC strains could be resolved at the RNA and protein level through differences in the sequence of a common RDase (DET1545-like homologs) and differences in expression of their vinyl chloride-respiring RDases. The dominant strain expresses VcrA, whereas the minor strain utilizes BvcA. We then used quantitative reverse-transcriptase PCR (qRT-PCR) as a targeted approach for quantifying transcript copies in the KB-1 TM consortium operated under a range of TCE respiration rates in continuously-fed, pseudo-steady-state reactors. These candidate biomarkers from KB-1 TM demonstrated a variety of trends in terms of transcript abundance as a function of respiration rate over the range: 7.7 × 10 -12 to 5.9 × 10 -10 microelectron equivalents per cell per hour (μeeq/cell∙h). Power law trends were observed between the respiration rate and transcript abundance for the main DMC RDase (VcrA) and the hydrogenase HupL (R² = 0.83 and 0.88, respectively), but not transcripts for 16S rRNA or three other RDases examined: TceA, BvcA or the RDase DET1545 homologs in KB1 TM . Overall, HupL transcripts appear to be the most robust activity biomarker across multiple DMC strains and in mixed communities including DMC co-cultures such as KB1 TM . The addition of oxygen induced cell stress that caused respiration rates

  15. Captodiamine, a putative antidepressant, enhances hypothalamic BDNF expression in vivo by synergistic 5-HT2c receptor antagonism and sigma-1 receptor agonism.

    Science.gov (United States)

    Ring, Rebecca M; Regan, Ciaran M

    2013-10-01

    The putative antidepressant captodiamine is a 5-HT2c receptor antagonist and agonist at sigma-1 and D3 dopamine receptors, exerts an anti-immobility action in the forced swim paradigm, and enhances dopamine turnover in the frontal cortex. Captodiamine has also been found to ameliorate stress-induced anhedonia, reduce the associated elevations of hypothalamic corticotrophin-releasing factor (CRF) and restore the reductions in hypothalamic BDNF expression. Here we demonstrate chronic administration of captodiamine to have no significant effect on hypothalamic CRF expression through sigma-1 receptor agonism; however, both sigma-1 receptor agonism or 5-HT2c receptor antagonism were necessary to enhance BDNF expression. Regulation of BDNF expression by captodiamine was associated with increased phosphorylation of transcription factor CREB and mediated through sigma-1 receptor agonism but blocked by 5-HT2c receptor antagonism. The existence of two separate signalling pathways was confirmed by immunolocalisation of each receptor to distinct cell populations in the paraventricular nucleus of the hypothalamus. Increased BDNF induced by captodiamine was also associated with enhanced expression of synapsin, but not PSD-95, suggesting induction of long-term structural plasticity between hypothalamic synapses. These unique features of captodiamine may contribute to its ability to ameliorate stress-induced anhedonia as the hypothalamus plays a prominent role in regulating HPA axis activity.

  16. Cardiac autonomic imbalance by social stress in rodents: understanding putative biomarkers

    Directory of Open Access Journals (Sweden)

    Susan K Wood, Phd

    2014-08-01

    Full Text Available Exposure to stress or traumatic events can lead to the development of depression and anxiety disorders. In addition to the debilitating consequences on mental health, patients with psychiatric disorders also suffer from autonomic imbalance, making them susceptible to a variety of medical disorders. Emerging evidence utilizing spectral analysis of heart rate variability (HRV, a reliable noninvasive measure of cardiovascular autonomic regulation, indicates that patients with depression and various anxiety disorders (i.e., panic, social, generalized anxiety disorders, and post traumatic stress disorder are characterized by decreased HRV. Social stressors in rodents are ethologically relevant experimental stressors that recapitulate many of the dysfunctional behavioral and physiological changes that occur in psychological disorders. In this review, evidence from clinical studies and preclinical stress models identify putative biomarkers capable of precipitating the comorbidity between disorders of the mind and autonomic dysfunction. Specifically, the role of corticotropin releasing factor, neuropeptide Y and inflammation are investigated. The impetus for this review is to highlight stress-related biomarkers that may prove critical in the development of autonomic imbalance in stress -related psychiatric disorders.

  17. Reduction redux.

    Science.gov (United States)

    Shapiro, Lawrence

    2018-04-01

    Putnam's criticisms of the identity theory attack a straw man. Fodor's criticisms of reduction attack a straw man. Properly interpreted, Nagel offered a conception of reduction that captures everything a physicalist could want. I update Nagel, introducing the idea of overlap, and show why multiple realization poses no challenge to reduction so construed. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. A putative non-hr origin of DNA replication in the HindIII-K fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus

    NARCIS (Netherlands)

    Kool, M.; Goldbach, R. W.; Vlak, J. M.

    1994-01-01

    In addition to the seven known homologous regions (hrs) of Autographa californica multiple nucleocapsid polyhedrosis virus (AcMNPV) the HindIII-K fragment was also found to carry a putative ori, although this fragment does not contain an hr. Deletion analysis showed that this ori contains several

  19. Screening for Genes Coding for Putative Antitumor Compounds, Antimicrobial and Enzymatic Activities from Haloalkalitolerant and Haloalkaliphilic Bacteria Strains of Algerian Sahara Soils

    Directory of Open Access Journals (Sweden)

    Okba Selama

    2014-01-01

    Full Text Available Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

  20. Purification, crystallization and preliminary X-ray diffraction studies of a putative UDP-N-acetyl-d-mannosamine dehydrogenase from Pyrococcus horikoshii OT3

    Energy Technology Data Exchange (ETDEWEB)

    Lokanath, Neratur K.; Pampa, Kudigana J.; Kamiya, Toshimi; Kunishima, Naoki, E-mail: kunisima@spring8.or.jp [Advanced Protein Crystallography Research Group, RIKEN SPring-8 Center, Harima Institute, 1-1-1 Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148 (Japan)

    2007-05-01

    A putative UDP-N-acetyl-d-mannosamine dehydrogenase from P. horikoshii OT3 has been crystallized in space group P2{sub 1}, with unit-cell parameters a = 80.28, b = 69.24, c = 83.10 Å, β = 114.4°. X-ray diffraction data have been collected to 1.80 Å resolution. A putative UDP-N-acetyl-d-mannosamine dehydrogenase from Pyrococcus horikoshii OT3, an essential enzyme for polysaccharide biosynthesis, has been overexpressed in Escherichia coli and purified. Crystals were obtained using the oil-microbatch method at 291 K. A native data set extending to 1.8 Å resolution has been collected and processed in space group P2{sub 1}. Assuming the presence of a dimer in the asymmetric unit, the V{sub M} value is calculated to be 2.3 Å{sup 3} Da{sup −1}, which is consistent with the result of a dynamic light-scattering experiment that shows a dimeric state of the protein in solution.

  1. Comparative Transcriptome Analysis Identifies Putative Genes Involved in the Biosynthesis of Xanthanolides in Xanthium strumarium L.

    Directory of Open Access Journals (Sweden)

    Yuanjun Li

    2016-08-01

    Full Text Available Xanthium strumarium L. is a traditional Chinese herb belonging to the Asteraceae family. The major bioactive components of this plant are sesquiterpene lactones, which include the xanthanolides. To date, the biogenesis of xanthanolides, especiallytheir downstream pathway, remains largely unknown. In X. strumarium, xanthanolides primarily accumulate in its glandular trichomes. To identify putative gene candidates involved in the biosynthesis of xanthanolides, three X. strumarium transcriptomes, which were derived from the young leaves of two different cultivars and the purified glandular trichomes from one of the cultivars, were constructed in this study. In total, 157 million clean reads were generated and assembled into 91,861 unigenes, of which 59,858 unigenes were successfully annotated. All the genes coding for known enzymes in the upstream pathway to the biosynthesis of xanthanolides were present in the X. strumarium transcriptomes. From a comparative analysis of the X. strumarium transcriptomes, this study identified a number of gene candidates that are putatively involved in the downstream pathway to the synthesis of xanthanolides, such as four unigenes encoding CYP71 P450s, 50 unigenes for dehydrogenases, and 27 genes for acetyltransferases. The possible functions of these four CYP71 candidates are extensively discussed. In addition, 116 transcription factors that were highly expressed in X. strumarium glandular trichomes were also identified. Their possible regulatory roles in the biosynthesis of sesquiterpene lactones are discussed. The global transcriptomic data for X. strumarium should provide a valuable resource for further research into the biosynthesis of xanthanolides.

  2. Humoral immune response to hypervariable region 1 of the putative envelope glycoprotein (gp70) of hepatitis C virus.

    OpenAIRE

    Kato, N; Sekiya, H; Ootsuyama, Y; Nakazawa, T; Hijikata, M; Ohkoshi, S; Shimotohno, K

    1993-01-01

    We recently found that alterations of amino acids in hypervariable region 1 (HVR1) of the putative envelope glycoprotein (gp70) of hepatitis C virus (HCV) occurred sequentially in the chronic phase of hepatitis at intervals of several months. This finding suggests that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection involving escape from the immunosurveillance system. To explore this possibility, we examined the humoral immune response to HVR1 with our assa...

  3. Analysis of the expression of putatively imprinted genes in bovine peri-implantation embryos

    DEFF Research Database (Denmark)

    Tveden-Nyborg, Pernille Yde; Alexopoulos, N.I.; Cooney, M.A.

    2008-01-01

    The application of assisted reproductive technologies (ART) has been shown to induce changes in the methylation of the embryonic genome, leading to aberrant gene expression, including that of imprinted genes. Aberrant methylation and gene expression has been linked to the large offspring syndrome...... (LOS) in bovine embryos resulting in increased embryonic morbidity and mortality. In the bovine, limited numbers of imprinted genes have been studied and studies have primarily been restricted to pre-implantation stages. This study reports original data on the expression pattern of 8 putatively...... imprinted genes (Ata3, Dlk1, Gnas, Grb10, Magel2, Mest-1, Ndn and Sgce) in bovine peri-implantation embryos. Two embryonic developmental stages were examined, Day 14 and Day 21. The gene expression pattern of single embryos was recorded for in vivo, in vitro produced (IVP) and parthenogenetic embryos...

  4. Evolutionary analysis of Pinus densata Masters, a putative Tertiary hybrid : 1. Allozyme variation.

    Science.gov (United States)

    Wang, X R; Szmidt, A E; Lewandowski, A; Wang, Z R

    1990-11-01

    Allozyme differentiation at 13 loci was studied in populations of Pinus tabulaeformis, P. densata, and P. yunnanensis from China. It was previously suggested that P. densata represents a Tertiary hybrid between P. tabulaeformis and P. yunnanensis. The observed levels of allozyme variation within and among the investigated species were comparable to those of other conifers. P. tabulaeformis differed markedly from P. yunnanensis with respect to allozyme frequencies, while P. densata was intermediate between the two putative parents. There was evidence of homozygote excess in embryos from all investigated species, as compared to Hardy-Weinberg expectations. The observed allozyme composition of P. densata conformed to earlier morphological and molecular evidence indicating hybrid origin of this taxon. It was proposed that fusion of gene pools from P. tabulaeformis and P. yunnanensis has led to adaptive evolution of a new species, P. densata.

  5. Functional Characterization of PaLAX1, a Putative Auxin Permease, in Heterologous Plant Systems1[W][OA

    Science.gov (United States)

    Hoyerová, Klára; Perry, Lucie; Hand, Paul; Laňková, Martina; Kocábek, Tomáš; May, Sean; Kottová, Jana; Pačes, Jan; Napier, Richard; Zažímalová, Eva

    2008-01-01

    We have isolated the cDNA of the gene PaLAX1 from a wild cherry tree (Prunus avium). The gene and its product are highly similar in sequences to both the cDNAs and the corresponding protein products of AUX/LAX-type genes, coding for putative auxin influx carriers. We have prepared and characterized transformed Nicotiana tabacum and Arabidopsis thaliana plants carrying the gene PaLAX1. We have proved that constitutive overexpression of PaLAX1 is accompanied by changes in the content and distribution of free indole-3-acetic acid, the major endogenous auxin. The increase in free indole-3-acetic acid content in transgenic plants resulted in various phenotype changes, typical for the auxin-overproducing plants. The uptake of synthetic auxin, 2,4-dichlorophenoxyacetic acid, was 3 times higher in transgenic lines compared to the wild-type lines and the treatment with the auxin uptake inhibitor 1-naphthoxyacetic acid reverted the changes caused by the expression of PaLAX1. Moreover, the agravitropic response could be restored by expression of PaLAX1 in the mutant aux1 plants, which are deficient in auxin influx carrier activity. Based on our data, we have concluded that the product of the gene PaLAX1 promotes the uptake of auxin into cells, and, as a putative auxin influx carrier, it affects the content and distribution of free endogenous auxin in transgenic plants. PMID:18184737

  6. Fluorescence-based biosensor for monitoring of environmental pollutants: From concept to field application

    Czech Academy of Sciences Publication Activity Database

    Bidmanová, Š.; Kotlanova, M.; Rataj, Tomáš; Damborský, J.; Trtílek, M.; Prokop, Z.

    2016-01-01

    Roč. 84, oct (2016), s. 97-105 ISSN 0956-5663 Grant - others:GA MŠk(CZ) LO1214 Institutional support: RVO:67179843 Keywords : dehydrochlorinase * environmental monitoring * field-testing * haloalkane dehalogenase * Halogenated pollutant * optical biosensor Subject RIV: EH - Ecology, Behaviour Impact factor: 7.780, year: 2016

  7. Haloalkane-utilizing Rhodococcus strains isolated from geographically distinct locations possess a highly conserved gene cluster encoding haloalkane catabolism

    NARCIS (Netherlands)

    Poelarends, GJ; Bosma, T; Kulakov, LA; Larkin, MJ; Marchesi, [No Value; Weightman, AJ; Janssen, DB; Kulakov, Leonid A.; Larkin, Michael J.; Marchesi, Julian R.; Weightman, Andrew J.

    The sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacterium Rhodococcus sp. strain NCIMB13064 were compared.

  8. Overproduction, crystallization and preliminary X-ray analysis of the putative l-ascorbate-6-phosphate lactonase UlaG from Escherichia coli

    International Nuclear Information System (INIS)

    Garces, Fernando; Fernández, Francisco J.; Pérez-Luque, Rosa; Aguilar, Juan; Baldomà, Laura; Coll, Miquel; Badía, Josefa; Vega, M. Cristina

    2007-01-01

    UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in E. coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized in a monoclinic space group. Crystals were obtained by the sitting-drop vapour-diffusion method at 293 K. A data set diffracting to 3 Å resolution was collected from a single crystal at 100 K. UlaG, the putative l-ascorbate-6-phosphate lactonase encoded by the ulaG gene from the utilization of l-ascorbate regulon in Escherichia coli, has been cloned, overexpressed, purified using standard chromatographic techniques and crystallized. Crystals were obtained by sitting-drop vapour diffusion at 293 K. Preliminary X-ray diffraction analysis revealed that the UlaG crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 104.52, b = 180.69, c = 112.88 Å, β = 103.26°. The asymmetric unit is expected to contain six copies of UlaG, with a corresponding volume per protein weight of 2.16 Å 3 Da −1 and a solvent content of 43%

  9. The crystal structure of human protein α1M reveals a chromophore-binding site and two putative protein–protein interfaces

    International Nuclear Information System (INIS)

    Zhang, Yangli; Gao, Zengqiang; Guo, Zhen; Zhang, Hongpeng; Zhang, Zhenzhen; Luo, Miao; Hou, Haifeng; Huang, Ailong; Dong, Yuhui; Wang, Deqiang

    2013-01-01

    Highlights: •We determined the first structure of human α1M with heavy electron density of the chromophore. •We proposed a new structural model of the chromophore. •We first revealed that the two conserved surface regions of α1M are proposed as putative protein–protein interface sites. -- Abstract: Lipocalin α1-microglobulin (α1M) is a conserved glycoprotein present in plasma and in the interstitial fluids of all tissues. α1M is linked to a heterogeneous yellow–brown chromophore of unknown structure, and interacts with several target proteins, including α1-inhibitor-3, fibronectin, prothrombin and albumin. To date, there is little knowledge about the interaction sites between α1M and its partners. Here, we report the crystal structure of the human α1M. Due to the crystallization occurring in a low ionic strength solution, the unidentified chromophore with heavy electron density is observed at a hydrophobic inner tube of α1M. In addition, two conserved surface regions of α1M are proposed as putative protein–protein interface sites. Further study is needed to unravel the detailed information about the interaction between α1M and its partners

  10. Sequence analysis and gene expression of putative exo- and endo-glucanases from oil palm (Elaeis guineensis) during fungal infection.

    Science.gov (United States)

    Yeoh, Keat-Ai; Othman, Abrizah; Meon, Sariah; Abdullah, Faridah; Ho, Chai-Ling

    2012-10-15

    Glucanases are enzymes that hydrolyze a variety β-d-glucosidic linkages. Plant β-1,3-glucanases are able to degrade fungal cell walls; and promote the release of cell-wall derived fungal elicitors. In this study, three full-length cDNA sequences encoding oil palm (Elaeis guineensis) glucanases were analyzed. Sequence analyses of the cDNA sequences suggested that EgGlc1-1 is a putative β-d-glucan exohydolase belonging to glycosyl hydrolase (GH) family 3 while EgGlc5-1 and EgGlc5-2 are putative glucan endo-1,3-β-glucosidases belonging to GH family 17. The transcript abundance of these genes in the roots and leaves of oil palm seedlings treated with Ganoderma boninense and Trichoderma harzianum was profiled to investigate the involvement of these glucanases in oil palm during fungal infection. The gene expression of EgGlc1-1 in the root of oil palm seedlings was increased by T. harzianum but suppressed by G. boninense; while the gene expression of both EgGlc5-1 and EgGlc5-2 in the roots of oil palm seedlings was suppressed by G. boninense or/and T. harzianum. Copyright © 2012 Elsevier GmbH. All rights reserved.

  11. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Bechard Matthew E.

    2003-01-01

    Full Text Available Tetrahydromethanopterin (H4MPT is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase. Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H4MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  12. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli.

    Science.gov (United States)

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H(4)MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H(4)MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H(4)MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H(4)MPT biosynthesis, the identification of putative RFAP synthase genes and measurement of RFAP synthase activity would provide an indication of the presence of H(4)MPT in untested microorganisms. Investigation of putative archaeal RFAP synthase genes has been hampered by the tendency of the resulting proteins to form inactive inclusion bodies in Escherichia coli. The current work describes a colorimetric assay for measuring RFAP synthase activity, and two modified procedures for expressing recombinant RFAP synthase genes to produce soluble, active enzyme. By lowering the incubation temperature during expression, RFAP synthase from Archaeoglobus fulgidus was produced in E. coli and purified to homogeneity. The production of active RFAP synthase from Methanothermobacter thermautotrophicus was achieved by coexpression of the gene MTH0830 with a molecular chaperone. This is the first direct biochemical identification of a methanogen gene that codes for an active RFAP synthase.

  13. Biominerlization and possible endosulfan degradation pathway adapted by Aspergillus niger.

    Science.gov (United States)

    Bhalerao, Tejomyee S

    2013-11-28

    Endosulfan is a chlorinated pesticide; its persistence in the environment and toxic effects on biota are demanding its removal. This study aims at improving the tolerance of the previously isolated fungus Aspergillus niger (A. niger) ARIFCC 1053 to endosulfan. Released chloride, dehalogenase activity, and released proteins were estimated along with analysis of endosulfan degradation and pathway identification. The culture could tolerate 1,000 mg/ml of technical grade endosulfan. Complete disappearance of endosulfan was seen after 168 h of incubation. The degradation study could easily be correlated with increase in released chlorides, dehalogenase activity and protein released. Comparative infrared spectral analysis suggested that the molecule of endosulfan was degraded efficiently by A. niger ARIFCC 1053. Obtained mass ion values by GC-MS suggested a hypothetical pathway during endosulfan degradation by A. niger ARIFCC 1053. All these results provide a basis for the development of bioremediation strategies to remediate the pollutant under study in the environment.

  14. Comparative analysis of the predicted secretomes of Rosaceae scab pathogens Venturia inaequalis and V. pirina reveals expanded effector families and putative determinants of host range.

    Science.gov (United States)

    Deng, Cecilia H; Plummer, Kim M; Jones, Darcy A B; Mesarich, Carl H; Shiller, Jason; Taranto, Adam P; Robinson, Andrew J; Kastner, Patrick; Hall, Nathan E; Templeton, Matthew D; Bowen, Joanna K

    2017-05-02

    Fungal plant pathogens belonging to the genus Venturia cause damaging scab diseases of members of the Rosaceae. In terms of economic impact, the most important of these are V. inaequalis, which infects apple, and V. pirina, which is a pathogen of European pear. Given that Venturia fungi colonise the sub-cuticular space without penetrating plant cells, it is assumed that effectors that contribute to virulence and determination of host range will be secreted into this plant-pathogen interface. Thus the predicted secretomes of a range of isolates of Venturia with distinct host-ranges were interrogated to reveal putative proteins involved in virulence and pathogenicity. Genomes of Venturia pirina (one European pear scab isolate) and Venturia inaequalis (three apple scab, and one loquat scab, isolates) were sequenced and the predicted secretomes of each isolate identified. RNA-Seq was conducted on the apple-specific V. inaequalis isolate Vi1 (in vitro and infected apple leaves) to highlight virulence and pathogenicity components of the secretome. Genes encoding over 600 small secreted proteins (candidate effectors) were identified, most of which are novel to Venturia, with expansion of putative effector families a feature of the genus. Numerous genes with similarity to Leptosphaeria maculans AvrLm6 and the Verticillium spp. Ave1 were identified. Candidates for avirulence effectors with cognate resistance genes involved in race-cultivar specificity were identified, as were putative proteins involved in host-species determination. Candidate effectors were found, on average, to be in regions of relatively low gene-density and in closer proximity to repeats (e.g. transposable elements), compared with core eukaryotic genes. Comparative secretomics has revealed candidate effectors from Venturia fungal plant pathogens that attack pome fruit. Effectors that are putative determinants of host range were identified; both those that may be involved in race-cultivar and host

  15. The inducible CAM plants in putative lunar lander experiments

    Science.gov (United States)

    Burlak, Olexii; Zaetz, Iryna; Soldatkin, Olexii; Rogutskyy, Ivan; Danilchenko, Boris; Mikheev, Olexander; de Vera, Jean-Pierre; Vidmachenko, Anatolii; Foing, Bernard H.; Kozyrovska, Natalia

    Precursory lunar lander experiments on growing plants in locker-based chambers will increase our understanding of effect of lunar conditions on plant physiology. The inducible CAM (Cras-sulacean Acid Metabolism)-plants are reasonable model for a study of relationships between environmental challenges and changes in plant/bacteria gene expression. In inducible CAM-plants the enzymatic machinery for the environmentally activated CAM switches on from a C3-to a full-CAM mode of photosynthesis in response to any stresses (Winter et al., 2008). In our study, Kalanchoe spp. are shown to be promising candidates for putative lunar experiments as resistant to irradiation and desiccation, especially after inoculation with a bacterial consortium (Boorlak et al., 2010). Within frames of the experiment we expect to get information about the functional activity of CAM-plants, in particular, its organogenesis, photosystem, the circadian regulation of plant metabolism on the base of data gaining with instrumental indications from expression of the reporter genes fused to any genes involved in vital functions of the plant (Kozyrovska et al., 2009). References 1. Winter K., Garcia M., Holtum J. (2008) J. Exp. Bot. 59(7):1829-1840 2. Bourlak O., Lar O., Rogutskyy I., Mikheev A., Zaets I., Chervatyuk N., de Vera J.-P., Danilchenko A.B. Foing B.H., zyrovska N. (2010) Space Sci. Technol. 3. Kozyrovska N.O., Vidmachenko A.P., Foing B.H. et al. Exploration/call/estec/ESA. 2009.

  16. Application of a Colorimetric Assay to Identify Putative Ribofuranosylaminobenzene 5'-Phosphate Synthase Genes Expressed with Activity in Escherichia coli

    OpenAIRE

    Bechard, Matthew E.; Chhatwal, Sonya; Garcia, Rosemarie E.; Rasche, Madeline E.

    2003-01-01

    Tetrahydromethanopterin (H4MPT) is a tetrahydrofolate analog originally discovered in methanogenic archaea, but later found in other archaea and bacteria. The extent to which H4MPT occurs among living organisms is unknown. The key enzyme which distinguishes the biosynthetic pathways of H4MPT and tetrahydrofolate is ribofuranosylaminobenzene 5'-phosphate synthase (RFAP synthase). Given the importance of RFAP synthase in H4MPT biosynthesis, the identification of putative RFAP synthase genes and...

  17. The putative serine protease inhibitor Api m 6 from Apis mellifera venom: recombinant and structural evaluation.

    Science.gov (United States)

    Michel, Y; McIntyre, M; Ginglinger, H; Ollert, M; Cifuentes, L; Blank, S; Spillner, E

    2012-01-01

    Immunoglobulin (Ig) E-mediated reactions to honeybee venom can cause severe anaphylaxis, sometimes with fatal consequences. Detailed knowledge of the allergic potential of all venom components is necessary to ensure proper diagnosis and treatment of allergy and to gain a better understanding of the allergological mechanisms of insect venoms. Our objective was to undertake an immunochemical and structural evaluation of the putative low-molecular-weight serine protease inhibitor Api m 6, a component of honeybee venom. We recombinantly produced Api m 6 as a soluble protein in Escherichia coli and in Spodoptera frugiperda (Sf9) insect cells.We also assessed specific IgE reactivity of venom-sensitized patients with 2 prokaryotically produced Api m 6 variants using enzyme-linked immunosorbent assay. Moreover, we built a structural model ofApi m 6 and compared it with other protease inhibitor structures to gain insights into the function of Api m 6. In a population of 31 honeybee venom-allergic patients, 26% showed specific IgE reactivity with prokaryotically produced Api m 6, showing it to be a minor but relevant allergen. Molecular modeling of Api m 6 revealed a typical fold of canonical protease inhibitors, supporting the putative function of this venom allergen. Although Api m 6 has a highly variant surface charge, its epitope distribution appears to be similar to that of related proteins. Api m 6 is a honeybee venom component with IgE-sensitizing potential in a fraction of venom-allergic patients. Recombinant Api m 6 can help elucidate individual component-resolved reactivity profiles and increase our understanding of immune responses to low-molecular-weight allergens

  18. Expression of Pluripotency and Oocyte-Related Genes in Single Putative Stem Cells from Human Adult Ovarian Surface Epithelium Cultured In Vitro in the Presence of Follicular Fluid

    Directory of Open Access Journals (Sweden)

    Irma Virant-Klun

    2013-01-01

    Full Text Available The aim of this study was to trigger the expression of genes related to oocytes in putative ovarian stem cells scraped from the ovarian surface epithelium of women with premature ovarian failure and cultured in vitro in the presence of follicular fluid, rich in substances for oocyte growth and maturation. Ovarian surface epithelium was scraped and cell cultures were set up by scrapings in five women with nonfunctional ovaries and with no naturally present mature follicles or oocytes. In the presence of donated follicular fluid putative stem cells grew and developed into primitive oocyte-like cells. A detailed single-cell gene expression profiling was performed to elucidate their genetic status in comparison to human embryonic stem cells, oocytes, and somatic fibroblasts. The ovarian cell cultures depleted/converted reproductive hormones from the culture medium. Estradiol alone or together with other substances may be involved in development of these primitive oocyte-like cells. The majority of primitive oocyte-like cells was mononuclear and expressed several genes related to pluripotency and oocytes, including genes related to meiosis, although they did not express some important oocyte-specific genes. Our work reveals the presence of putative stem cells in the ovarian surface epithelium of women with premature ovarian failure.

  19. Identification of a novel group of putative Arabidopsis thaliana beta-(1,3)-galactosyltransferases

    DEFF Research Database (Denmark)

    Qu, Yongmei; Egelund, Jack; Gilson, Paul R

    2008-01-01

    To begin biochemical and molecular studies on the biosynthesis of the type II arabinogalactan chains on arabinogalactan-proteins (AGPs), we adopted a bioinformatic approach to identify and systematically characterise the putative galactosyltransferases (GalTs) responsible for synthesizing the beta......-(1,3)-Gal linkage from CAZy GT-family-31 from Arabidopsis thaliana. These analyses confirmed that 20 members of the GT-31 family contained domains/motifs typical of biochemically characterised beta-(1,3)-GTs from mammalian systems. Microarray data confirm that members of this family are expressed......,3)-GalT activity. This bioinformatic/molecular study of CAZy GT-family-31 was validated by the recent report of Strasser et al. (Plant Cell 19:2278-2292, 2007) that another member of this family (At1g26810; GALT1) encodes a beta-(1,3)-GalT involved in the biosynthesis of the Lewis a epitope of N...

  20. Complete Genome Sequence of Diaphorina citri-associated C virus, a Novel Putative RNA Virus of the Asian Citrus Psyllid, Diaphorina citri

    OpenAIRE

    Nouri, Shahideh; Salem, Nid?; Falk, Bryce W.

    2016-01-01

    We present here the complete nucleotide sequence and genome organization of a novel putative RNA virus identified in field populations of the Asian citrus psyllid, Diaphorina citri, through sequencing of the transcriptome followed by reverse transcription-PCR (RT-PCR). We tentatively named this virus Diaphorina citri-associated C virus (DcACV). DcACV is an unclassified positive-sense RNA virus.

  1. Microbial reductive dehalogenation.

    Science.gov (United States)

    Mohn, W W; Tiedje, J M

    1992-01-01

    A wide variety of compounds can be biodegraded via reductive removal of halogen substituents. This process can degrade toxic pollutants, some of which are not known to be biodegraded by any other means. Reductive dehalogenation of aromatic compounds has been found primarily in undefined, syntrophic anaerobic communities. We discuss ecological and physiological principles which appear to be important in these communities and evaluate how widely applicable these principles are. Anaerobic communities that catalyze reductive dehalogenation appear to differ in many respects. A large number of pure cultures which catalyze reductive dehalogenation of aliphatic compounds are known, in contrast to only a few organisms which catalyze reductive dehalogenation of aromatic compounds. Desulfomonile tiedjei DCB-1 is an anaerobe which dehalogenates aromatic compounds and is physiologically and morphologically unusual in a number of respects, including the ability to exploit reductive dehalogenation for energy metabolism. When possible, we use D. tiedjei as a model to understand dehalogenating organisms in the above-mentioned undefined systems. Aerobes use reductive dehalogenation for substrates which are resistant to known mechanisms of oxidative attack. Reductive dehalogenation, especially of aliphatic compounds, has recently been found in cell-free systems. These systems give us an insight into how and why microorganisms catalyze this activity. In some cases transition metal complexes serve as catalysts, whereas in other cases, particularly with aromatic substrates, the catalysts appear to be enzymes. Images PMID:1406492

  2. Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma)

    DEFF Research Database (Denmark)

    Tipsmark, Christian K; Luckenbach, J Adam; Madsen, Steffen S

    2008-01-01

    The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotrans...... process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates....

  3. Enema reduction of intussusception: the success rate of hydrostatic and pneumatic reduction.

    Science.gov (United States)

    Khorana, Jiraporn; Singhavejsakul, Jesda; Ukarapol, Nuthapong; Laohapensang, Mongkol; Wakhanrittee, Junsujee; Patumanond, Jayanton

    2015-01-01

    Intussusception is a common surgical emergency in infants and children. The incidence of intussusception is from one to four per 2,000 infants and children. If there is no peritonitis, perforation sign on abdominal radiographic studies, and nonresponsive shock, nonoperative reduction by pneumatic or hydrostatic enema can be performed. The purpose of this study was to compare the success rates of both the methods. Two institutional retrospective cohort studies were performed. All intussusception patients (ICD-10 code K56.1) who had visited Chiang Mai University Hospital and Siriraj Hospital from January 2006 to December 2012 were included in the study. The data were obtained by chart reviews and electronic databases, which included demographic data, symptoms, signs, and investigations. The patients were grouped according to the method of reduction followed into pneumatic reduction and hydrostatic reduction groups with the outcome being the success of the reduction technique. One hundred and seventy episodes of intussusception occurring in the patients of Chiang Mai University Hospital and Siriraj Hospital were included in this study. The success rate of pneumatic reduction was 61% and that of hydrostatic reduction was 44% (P=0.036). Multivariable analysis and adjusting of the factors by propensity scores were performed; the success rate of pneumatic reduction was 1.48 times more than that of hydrostatic reduction (P=0.036, 95% confidence interval [CI] =1.03-2.13). Both pneumatic and hydrostatic reduction can be performed safely according to the experience of the radiologist or pediatric surgeon and hospital setting. This study showed that pneumatic reduction had a higher success rate than hydrostatic reduction.

  4. Conformational changes associated with the binding of zinc acetate at the putative active site of XcTcmJ, a cupin from Xanthomonas campestris pv. campestris

    International Nuclear Information System (INIS)

    Axelrod, Herbert L.; Kozbial, Piotr; McMullan, Daniel; Krishna, S. Sri; Miller, Mitchell D.; Abdubek, Polat; Acosta, Claire; Astakhova, Tamara; Carlton, Dennis; Caruthers, Jonathan; Chiu, Hsiu-Ju; Clayton, Thomas; Deller, Marc C.; Duan, Lian; Elias, Ylva; Feuerhelm, Julie; Grzechnik, Slawomir K.; Grant, Joanna C.; Han, Gye Won; Jaroszewski, Lukasz; Jin, Kevin K.; Klock, Heath E.; Knuth, Mark W.; Kumar, Abhinav; Marciano, David; Morse, Andrew T.; Murphy, Kevin D.; Nigoghossian, Edward; Okach, Linda; Oommachen, Silvya; Paulsen, Jessica; Reyes, Ron; Rife, Christopher L.; Tien, Henry J.; Trout, Christina V.; Bedem, Henry van den; Weekes, Dana; White, Aprilfawn; Xu, Qingping; Zubieta, Chloe; Hodgson, Keith O.; Wooley, John; Elsliger, Marc-André; Deacon, Ashley M.; Godzik, Adam; Lesley, Scott A.; Wilson, Ian A.

    2009-01-01

    The crystal structure of an RmlC-type cupin with zinc acetate bound at the putative active site reveals significant differences from a previous structure without any bound ligand. The functional implications of the ligand-induced conformational changes are discussed. In the plant pathogen Xanthomonas campestris pv. campestris, the product of the tcmJ gene, XcTcmJ, encodes a protein belonging to the RmlC family of cupins. XcTcmJ was crystallized in a monoclinic space group (C2) in the presence of zinc acetate and the structure was determined to 1.6 Å resolution. Previously, the apo structure has been reported in the absence of any bound metal ion [Chin et al. (2006 ▶), Proteins, 65, 1046–1050]. The most significant difference between the apo structure and the structure of XcTcmJ described here is a reorganization of the binding site for zinc acetate, which was most likely acquired from the crystallization solution. This site is located in the conserved metal ion-binding domain at the putative active site of XcTcmJ. In addition, an acetate was also bound within coordination distance of the zinc. In order to accommodate this binding, rearrangement of a conserved histidine ligand is required as well as several nearby residues within and around the putative active site. These observations indicate that binding of zinc serves a functional role in this cupin protein

  5. Expression, purification and DNA-binding activities of two putative ModE proteins of Herbaspirillum seropedicae (Burkholderiales, Oxalobacteraceae

    Directory of Open Access Journals (Sweden)

    André L.F. Souza

    2008-01-01

    Full Text Available In prokaryotes molybdenum is taken up by a high-affinity ABC-type transporter system encoded by the modABC genes. The endophyte β-Proteobacterium Herbaspirillum seropedicae has two modABC gene clusters and two genes encoding putative Mo-dependent regulator proteins (ModE1 and ModE2. Analysis of the amino acid sequence of the ModE1 protein of H. seropedicae revealed the presence of an N-terminal domain containing a DNA-binding helix-turn-helix motif (HTH and a C-terminal domain with a molybdate-binding motif. The second putative regulator protein, ModE2, contains only the helix-turn-helix motif, similar to that observed in some sequenced genomes. We cloned the modE1 (810 bp and modE2 (372 bp genes and expressed them in Escherichia coli as His-tagged fusion proteins, which we subsequently purified. The over-expressed recombinant His-ModE1 was insoluble and was purified after solubilization with urea and then on-column refolded during affinity chromatography. The His-ModE2 was expressed as a soluble protein and purified by affinity chromatography. These purified proteins were analyzed by DNA band-shift assays using the modA2 promoter region as probe. Our results indicate that His-ModE1 and His-ModE2 are able to bind to the modA2 promoter region, suggesting that both proteins may play a role in the regulation of molybdenum uptake and metabolism in H. seropedicae.

  6. Molecular analysis of mxbD and mxbM, a putative sensor-regulator pair required for oxidation of methanol in Methylobacterium extorquens AM1.

    Science.gov (United States)

    Springer, A L; Morris, C J; Lidstrom, M E

    1997-05-01

    Five genes are thought to be required for transcription of methanol oxidation genes in Methylobacterium strains. These putative regulatory genes include mxcQE, which encode a putative sensor-regulator pair, and mxbDM and mxaB, whose functions are less well-understood. In this study, mxbDM in Methylobacterium extorquens AM1 were shown to be required for expression of a xylE transcriptional fusion to the structural gene for the large subunit of methanol dehydrogenase (mxaF), confirming the role of these genes in transcriptional regulation of mxaF. The nucleotide sequence suggests that mxbD encodes a histidine protein kinase with two transmembrane domains and that mxbM encodes a DNA-binding response regulator. A xylE transcriptional fusion to the putative mxbD promoter showed low-level expression in wild-type cells grown on one-carbon (C1) compounds and no detectable expression in cells grown on succinate. Deletion analysis of this promoter construct showed that the region 229-129 bp upstream of the start of mxbD is required for expression. The expression of the mxbD-xylE fusion was examined in each of the five known regulatory mutant classes. xylE expression was reduced to non-detectable levels in MxcQ and MxcE mutants, but was not affected in the other regulatory mutants or in non-regulatory mutants defective in methanol oxidation. These results suggest a regulatory hierarchy in which the sensor-regulator pair MxcQE control expression of the sensor-regulator pair MxbDM, and MxbDM in turn control expression of a number of genes involved in methanol oxidation.

  7. Principal Components as a Data Reduction and Noise Reduction Technique

    Science.gov (United States)

    Imhoff, M. L.; Campbell, W. J.

    1982-01-01

    The potential of principal components as a pipeline data reduction technique for thematic mapper data was assessed and principal components analysis and its transformation as a noise reduction technique was examined. Two primary factors were considered: (1) how might data reduction and noise reduction using the principal components transformation affect the extraction of accurate spectral classifications; and (2) what are the real savings in terms of computer processing and storage costs of using reduced data over the full 7-band TM complement. An area in central Pennsylvania was chosen for a study area. The image data for the project were collected using the Earth Resources Laboratory's thematic mapper simulator (TMS) instrument.

  8. Transcriptome analysis of Spodoptera frugiperda Sf9 cells reveals putative apoptosis-related genes and a preliminary apoptosis mechanism induced by azadirachtin.

    Science.gov (United States)

    Shu, Benshui; Zhang, Jingjing; Sethuraman, Veeran; Cui, Gaofeng; Yi, Xin; Zhong, Guohua

    2017-10-16

    As an important botanical pesticide, azadirachtin demonstrates broad insecticidal activity against many agricultural pests. The results of a previous study indicated the toxicity and apoptosis induction of azadirachtin in Spodoptera frugiperda Sf9 cells. However, the lack of genomic data has hindered a deeper investigation of apoptosis in Sf9 cells at a molecular level. In the present study, the complete transcriptome data for Sf9 cell line was accomplished using Illumina sequencing technology, and 97 putative apoptosis-related genes were identified through BLAST and KEGG orthologue annotations. Fragments of potential candidate apoptosis-related genes were cloned, and the mRNA expression patterns of ten identified genes regulated by azadirachtin were examined using qRT-PCR. Furthermore, Western blot analysis showed that six putative apoptosis-related proteins were upregulated after being treated with azadirachtin while the protein Bcl-2 were downregulated. These data suggested that both intrinsic and extrinsic apoptotic signal pathways comprising the identified potential apoptosis-related genes were potentially active in S. frugiperda. In addition, the preliminary results revealed that caspase-dependent or caspase-independent apoptotic pathways could function in azadirachtin-induced apoptosis in Sf9 cells.

  9. Portal inflammation during NAFLD is frequent and associated with the early phases of putative hepatic progenitor cell activation.

    Science.gov (United States)

    Carotti, Simone; Vespasiani-Gentilucci, Umberto; Perrone, Giuseppe; Picardi, Antonio; Morini, Sergio

    2015-11-01

    We investigated whether portal tract inflammation observed in non-alcoholic fatty liver disease (NAFLD) is associated with hepatic progenitor cell compartment activation, as thoroughly evaluated with different markers of the staminal lineage. Fifty-two patients with NAFLD were studied. NAFLD activity score, fibrosis and portal inflammation were histologically evaluated. Putative hepatic progenitor cells, intermediate hepatobiliary cells and bile ductules/interlobular bile ducts were evaluated by immunohistochemistry for cytokeratin (CK)-7, CK-19 and epithelial cell adhesion molecule (EpCAM), and a hepatic progenitor cell compartment score was derived. Hepatic stellate cell and myofibroblast activity was determined by immunohistochemistry for α-smooth muscle actin. Portal inflammation was absent in a minority of patients, mild in 40% of cases and more than mild in about half of patients, showing a strong correlation with fibrosis (r=0.76, pcells (r=0.48, pcells (r=0.6, pcell compartment activation were associated with portal inflammation by univariate analysis. In the multivariate model, the only variable independently associated with portal inflammation was hepatic progenitor cell compartment activation (OR 3.7, 95% CI 1.1 to 12.6). Portal inflammation is frequent during NAFLD and strongly associated with activation of putative hepatic progenitor cells since the first steps of their differentiation, portal myofibroblast activity and fibrosis. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  10. Shell alterations in limpets as putative biomarkers for multi-impacted coastal areas.

    Science.gov (United States)

    Begliomini, Felipe Nincao; Maciel, Daniele Claudino; de Almeida, Sérgio Mendonça; Abessa, Denis Moledo; Maranho, Luciane Alves; Pereira, Camilo Seabra; Yogui, Gilvan Takeshi; Zanardi-Lamardo, Eliete; Castro, Ítalo Braga

    2017-07-01

    During the last years, shell alterations in gastropods have been proposed as tools to be used in monitoring programs. However, no studies were so far performed investigating the relationships among shell parameters and classical biomarkers of damage. The relationship between shell alterations (biometrics, shape and elemental composition) and biomarkers (LPO and DNA strand break) was evaluated in the limpet L. subrugosa sampled along a contamination gradient in a multi-impacted coastal zone from southeastern Brazil. Statistically significant differences were detected among sites under different pollution levels. The occurrence of shell malformations was consistent with environmental levels of several hazardous substances reported for the studied area and related to lipid peroxidation and DNA damage. In addition, considering the low mobility, wide geographic distribution, ease of collection and abundance of limpets in coastal zones, this putative tool may be a cost-effective alternative to traditional biomarkers. Thus, shell alterations in limpets seem to be good proxies for assessing biological adverse effects in multi-impacted coastal zones. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Data of expression status of miR- 29a and its putative target mitochondrial apoptosis regulatory gene DRP1 upon miR-15a and miR-214 inhibition

    Directory of Open Access Journals (Sweden)

    Muhammad Ishtiaq Jan

    2018-02-01

    Full Text Available Data is about the mitochondrial apoptosis regulatory framework genes PUMA, DRP1 (apoptotic, and ARC (anti-apoptotic analysis after the employment of their controlling miRNAs inhibitors. The data represents putative conserved targeting of seed regions of miR-15a, miR-29a, and miR-214 with respective target genes PUMA, DRP1, and ARC. Data is of cross interference in expression levels of one miRNA family, miR-29a and its putative target DRP1 upon the inhibitory treatment of other miRNAs 15a and 214. Keywords: DRP1, miR-15a, Apoptosis, miRNAs inhibition

  12. Reduction in language testing

    DEFF Research Database (Denmark)

    Dimova, Slobodanka; Jensen, Christian

    2013-01-01

    /video recorded speech samples and written reports produced by two experienced raters after testing. Our findings suggest that reduction or reduction-like pronunciation features are found in tested L2 speech, but whenever raters identify and comment on such reductions, they tend to assess reductions negatively......This study represents an initial exploration of raters' comments and actual realisations of form reductions in L2 test speech performances. Performances of three L2 speakers were selected as case studies and illustrations of how reductions are evaluated by the raters. The analysis is based on audio...

  13. Strigolactone-Induced Putative Secreted Protein 1 Is Required for the Establishment of Symbiosis by the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis.

    Science.gov (United States)

    Tsuzuki, Syusaku; Handa, Yoshihiro; Takeda, Naoya; Kawaguchi, Masayoshi

    2016-04-01

    Arbuscular mycorrhizal (AM) symbiosis is the most widespread association between plants and fungi. To provide novel insights into the molecular mechanisms of AM symbiosis, we screened and investigated genes of the AM fungus Rhizophagus irregularis that contribute to the infection of host plants. R. irregularis genes involved in the infection were explored by RNA-sequencing (RNA-seq) analysis. One of the identified genes was then characterized by a reverse genetic approach using host-induced gene silencing (HIGS), which causes RNA interference in the fungus via the host plant. The RNA-seq analysis revealed that 19 genes are up-regulated by both treatment with strigolactone (SL) (a plant symbiotic signal) and symbiosis. Eleven of the 19 genes were predicted to encode secreted proteins and, of these, SL-induced putative secreted protein 1 (SIS1) showed the largest induction under both conditions. In hairy roots of Medicago truncatula, SIS1 expression is knocked down by HIGS, resulting in significant suppression of colonization and formation of stunted arbuscules. These results suggest that SIS1 is a putative secreted protein that is induced in a wide spatiotemporal range including both the presymbiotic and symbiotic stages and that SIS1 positively regulates colonization of host plants by R. irregularis.

  14. PigZ, a TetR/AcrR family repressor, modulates secondary metabolism via the expression of a putative four-component resistance-nodulation-cell-division efflux pump, ZrpADBC, in Serratia sp. ATCC 39006.

    Science.gov (United States)

    Gristwood, Tamzin; Fineran, Peter C; Everson, Lee; Salmond, George P C

    2008-07-01

    The Gram-negative enterobacterium, Serratia sp. ATCC 39006 synthesizes several secondary metabolites, including prodigiosin (Pig) and a carbapenem antibiotic (Car). A complex hierarchical network of regulatory proteins control Pig and Car production. In this study we characterize a TetR family regulator, PigZ, which represses transcription of a divergently transcribed putative resistance-nodulation-cell-division (RND) efflux pump, encoded by zrp (PigZ repressed pump) ADBC, via direct binding to the zrpA-pigZ intergenic region. Unusually, this putative RND pump contains two predicted membrane fusion proteins (MFPs), ZrpA and ZrpD. A mutation in pigZ resulted in multiple phenotypic changes, including exoenzyme production, motility and differential regulation of Pig and Car production. A polar suppressor mutation, within zrpA, restored all tested phenotypes to parental strain levels, indicating that the changes observed are due to the increase in expression of ZrpADBC in the absence of the repressor, PigZ. Genomic deletions of zrpA and zrpD indicate that the MFP ZrpD, but not ZrpA, is essential for activity of the putative pump. Bioinformatic analysis revealed that putative RND efflux pumps encoding two MFP components are not uncommon, particularly among plant-associated, Gram-negative bacteria. In addition, based on phylogenetic analysis, we propose that these pairs of MFPs consist of two distinct subtypes.

  15. Process energy reduction

    International Nuclear Information System (INIS)

    Lowthian, W.E.

    1993-01-01

    Process Energy Reduction (PER) is a demand-side energy reduction approach which complements and often supplants other traditional energy reduction methods such as conservation and heat recovery. Because the application of PER is less obvious than the traditional methods, it takes some time to learn the steps as well as practice to become proficient in its use. However, the benefit is significant, often far outweighing the traditional energy reduction approaches. Furthermore, the method usually results in a better process having less waste and pollution along with improved yields, increased capacity, and lower operating costs

  16. Complete Genome Sequence of Diaphorina citri-associated C virus, a Novel Putative RNA Virus of the Asian Citrus Psyllid, Diaphorina citri.

    Science.gov (United States)

    Nouri, Shahideh; Salem, Nidà; Falk, Bryce W

    2016-07-21

    We present here the complete nucleotide sequence and genome organization of a novel putative RNA virus identified in field populations of the Asian citrus psyllid, Diaphorina citri, through sequencing of the transcriptome followed by reverse transcription-PCR (RT-PCR). We tentatively named this virus Diaphorina citri-associated C virus (DcACV). DcACV is an unclassified positive-sense RNA virus. Copyright © 2016 Nouri et al.

  17. Insights into the Cytoadherence Phenomenon of Plasmodium vivax: The Putative Role of Phosphatidylserine

    Directory of Open Access Journals (Sweden)

    Paulo Renato Totino

    2017-09-01

    Full Text Available Plasmodium vivax is the most geographically widespread and the dominant human malaria parasite in most countries outside of sub-Saharan Africa and, although it was classically recognized to cause benign infection, severe cases and deaths caused by P. vivax have remarkably been reported. In contrast to Plasmodium falciparum, which well-known ability to bind to endothelium and placental tissue and form rosettes is related to severity of the disease, it has been a dogma that P. vivax is unable to undergo cytoadherent phenomena. However, some studies have demonstrated that red blood cells (RBCs infected by P. vivax can cytoadhere to host cells, while the molecules participating in this host–parasite interaction are still a matter of speculation. In the present overview, we address the evidences currently supporting the adhesive profile of P. vivax and, additionally, discuss the putative role of phosphatidylserine—a cell membrane phospholipid with cytoadhesive properties that has been detected on the surface of Plasmodium-parasitized RBCs.

  18. Flavoprotein-mediated tellurite reduction: structural basis and applications to the synthesis of tellurium-containing nanostructures

    Directory of Open Access Journals (Sweden)

    Mauricio Arenas-Salinas

    2016-07-01

    Full Text Available The tellurium oxyanion tellurite (TeO32- is extremely harmful for most organisms. It has been suggested that a potential bacterial tellurite resistance mechanism would consist of an enzymatic, NAD(PH-dependent, reduction to the less toxic form elemental tellurium (Te0. To date, a number of enzymes such as catalase, type II NADH dehydrogenase and terminal oxidases from the electron transport chain, nitrate reductases, and dihydrolipoamide dehydrogenase (E3, among others, have been shown to display tellurite-reducing activity. This activity is generically referred to as tellurite reductase (TR. Bioinformatic data resting on some of the abovementioned enzymes enabled the identification of common structures involved in tellurite reduction including vicinal catalytic cysteine residues and the FAD/NAD(P+-binding domain, which is characteristic of some flavoproteins. Along this line, thioredoxin reductase (TrxB, alkyl hydroperoxide reductase (AhpF, glutathione reductase (GorA, mercuric reductase (MerA, NADH: flavorubredoxin reductase (NorW, dihydrolipoamide dehydrogenase, and the putative oxidoreductase YkgC from Escherichia coli or environmental bacteria were purified and assessed for TR activity. All of them displayed in vitro TR activity at the expense of NADH or NADPH oxidation. In general, optimal reducing conditions occurred around pH 9-10 and 37 °C.Enzymes exhibiting strong TR activity produced Te-containing nanostructures (TeNS. While GorA and AhpF generated TeNS of 75 nm average diameter, E3 and YkgC produced larger structures (> 100 nm. Electron-dense structures were observed in cells over-expressing genes encoding TrxB, GorA and YkgC.

  19. Characterization of a putative grapevine Zn transporter, VvZIP3, suggests its involvement in early reproductive development in Vitis vinifera L

    Directory of Open Access Journals (Sweden)

    Gainza-Cortés Felipe

    2012-07-01

    Full Text Available Abstract Background Zinc (Zn deficiency is one of the most widespread mineral nutritional problems that affect normal development in plants. Because Zn cannot passively diffuse across cell membranes, it must be transported into intracellular compartments for all biological processes where Zn is required. Several members of the Zinc-regulated transporters, Iron-regulated transporter-like Protein (ZIP gene family have been characterized in plants, and have shown to be involved in metal uptake and transport. This study describes the first putative Zn transporter in grapevine. Unravelling its function may explain an important symptom of Zn deficiency in grapevines, which is the production of clusters with fewer and usually smaller berries than normal. Results We identified and characterized a putative Zn transporter from berries of Vitis vinifera L., named VvZIP3. Compared to other members of the ZIP family identified in the Vitis vinifera L. genome, VvZIP3 is mainly expressed in reproductive tissue - specifically in developing flowers - which correlates with the high Zn accumulation in these organs. Contrary to this, the low expression of VvZIP3 in parthenocarpic berries shows a relationship with the lower Zn accumulation in this tissue than in normal seeded berries where its expression is induced by Zn. The predicted protein sequence indicates strong similarity with several members of the ZIP family from Arabidopsis thaliana and other species. Moreover, VvZIP3 complemented the growth defect of a yeast Zn-uptake mutant, ZHY3, and is localized in the plasma membrane of plant cells, suggesting that VvZIP3 has the function of a Zn uptake transporter. Conclusions Our results suggest that VvZIP3 encodes a putative plasma membrane Zn transporter protein member of the ZIP gene family that might play a role in Zn uptake and distribution during the early reproductive development in Vitis vinifera L., indicating that the availability of this micronutrient

  20. Direct Involvement of ombB, omaB and omcB Genes in Extracellular Reduction of Fe(III by Geobacter sulfurreducens PCA

    Directory of Open Access Journals (Sweden)

    Yimo eLiu

    2015-10-01

    Full Text Available The tandem gene clusters orfR-ombB-omaB-omcB and orfS-ombC-omaC-omcC of the metal-reducing bacterium Geobacter sulfurreducens PCA are responsible for trans-outer membrane electron transfer during extracellular reduction of Fe(III-citrate and ferrihydrite [a poorly crystalline Fe(III oxide]. Each gene cluster encodes a putative transcriptional factor (OrfR/OrfS, a porin-like outer-membrane protein (OmbB/OmbC, a periplasmic c-type cytochrome (c-Cyt, OmaB/OmaC and an outer-membrane c-Cyt (OmcB/OmcC. The individual roles of OmbB, OmaB and OmcB in extracellular reduction of Fe(III, however, have remained either uninvestigated or controversial. Here, we showed that replacements of ombB, omaB, omcB and ombB-omaB with an antibiotic gene in the presence of ombC-omaC-omcC had no impact on reduction of Fe(III-citrate by G. sulfurreducens PCA. Disruption of ombB, omaB, omcB and ombB-omaB in the absence of ombC-omaC-omcC, however, severely impaired the bacterial ability to reduce Fe(III-citrate as well as ferrihydrite. These results unequivocally demonstrate an overlapping role of ombB-omaB-omcB and ombC-omaC-omcC in extracellular Fe(III reduction by G. sulfurreducens PCA. Involvement of both ombB-omaB-omcB and ombC-omaC-omcC in extracellular Fe(III reduction reflects the importance of these trans-outer membrane protein complexes in the physiology of this bacterium. Moreover, the kinetics of Fe(III-citrate and ferrihydrite reduction by these mutants in the absence of ombC-omaC-omcC were nearly identical, which suggests that absence of any protein subunit eliminates function of OmaB/OmbB/OmcB protein complex. Finally, orfS was found to have a negative impact on the extracellular reduction of Fe(III-citrate and ferrihydrite in G. sulfurreducens PCA probably by serving as a transcriptional repressor.

  1. A systematic review of amyloid-beta peptides as putative mediators of the association between affective disorders and Alzheimer's disease

    DEFF Research Database (Denmark)

    Abbasowa, Leda; Heegaard, N. H. H.

    2014-01-01

    Background: Affective disorders are associated with an increased occurrence of cognitive deficits and have been linked to cognitive impairment and Alzheimer's disease. The putative molecular mechanisms involved in these associations are however not clear. The aim of this systematic review...... were limited by very low sample numbers. Finally, different assays for amyloid-beta were utilized in the different studies, thus hampering comparisons. Conclusion: To unravel possible risk relations and causalities between affective disorder and Alzheimer's disease and to determine how amyloid...

  2. Expression profiles of putative defence-related proteins in oil palm (Elaeis guineensis) colonized by Ganoderma boninense.

    Science.gov (United States)

    Tan, Yung-Chie; Yeoh, Keat-Ai; Wong, Mui-Yun; Ho, Chai-Ling

    2013-11-01

    Basal stem rot (BSR) is a major disease of oil palm caused by a pathogenic fungus, Ganoderma boninense. However, the interaction between the host plant and its pathogen is not well characterized. To better understand the response of oil palm to G. boninense, transcript profiles of eleven putative defence-related genes from oil palm were measured by quantitative reverse-transcription (qRT)-PCR in the roots of oil palms treated with G. boninense from 3 to 12 weeks post infection (wpi). These transcripts encode putative Bowman-Birk serine protease inhibitors (EgBBI1 and 2), defensin (EgDFS), dehydrin (EgDHN), early methionine-labeled polypeptides (EgEMLP1 and 2), glycine-rich RNA binding protein (EgGRRBP), isoflavone reductase (EgIFR), metallothionein-like protein (EgMT), pathogenesis-related-1 protein (EgPRP), and type 2 ribosome-inactivating protein (EgT2RIP). The transcript abundance of EgBBI2 increased in G. boninense-treated roots at 3 and 6wpi compared to those of controls; while the transcript abundance of EgBBI1, EgDFS, EgEMLP1, EgMT, and EgT2RIP increased in G. boninense-treated roots at 6 or 12wpi. Meanwhile, the gene expression of EgDHN was up-regulated at all three time points in G. boninense-treated roots. The expression profiles of the eleven transcripts were also studied in leaf samples upon inoculation of G. boninense and Trichoderma harzianum to identify potential biomarkers for early detection of BSR. Two candidate genes (EgEMLP1 and EgMT) that have different profiles in G. boninense-treated leaves compared to those infected by T. harzianum may have the potential to be developed as biomarkers for early detection of G. boninense infection. Copyright © 2013 Elsevier GmbH. All rights reserved.

  3. Determination of reduction yield of lithium metal reduction process

    International Nuclear Information System (INIS)

    Choi, In Kyu; Cho, Young Hwan; Kim, Taek Jin; Jee, Kwang Young

    2004-01-01

    Metal reduction of spent oxide fuel is the first step for the effective storage of spent fuel in Korea as well as transmutation purpose of long-lived radio-nuclides. During the reduction of uranium oxide by lithium metal to uranium metal, lithium oxide is stoichiometrically produced. By determining the concentration of lithium oxide in lithium chloride, we can estimate that how much uranium oxide is converted to uranium metal. Previous method to determine the lithium oxide concentration in lithium chloride is tedious and timing consuming. This paper describe the on-line monitoring method of lithium oxide during the reduction process

  4. Modern Reduction Methods

    CERN Document Server

    Andersson, Pher G

    2008-01-01

    With its comprehensive overview of modern reduction methods, this book features high quality contributions allowing readers to find reliable solutions quickly and easily. The monograph treats the reduction of carbonyles, alkenes, imines and alkynes, as well as reductive aminations and cross and heck couplings, before finishing off with sections on kinetic resolutions and hydrogenolysis. An indispensable lab companion for every chemist.

  5. PHTS, a novel putative tumor suppressor, is involved in the transformation reversion of HeLaHF cells independently of the p53 pathway

    International Nuclear Information System (INIS)

    Yu Dehua; Fan, Wufang; Liu, Guohong; Nguy, Vivian; Chatterton, Jon E.; Long Shilong; Ke, Ning; Meyhack, Bernd; Bruengger, Adrian; Brachat, Arndt; Wong-Staal, Flossie; Li, Qi-Xiang

    2006-01-01

    HeLaHF is a non-transformed revertant of HeLa cells, likely resulting from the activation of a putative tumor suppressor(s). p53 protein was stabilized in this revertant and reactivated for certain transactivation functions. Although p53 stabilization has not conclusively been linked to the reversion, it is clear that the genes in p53 pathway are involved. The present study confirms the direct role of p53 in HeLaHF reversion by demonstrating that RNAi-mediated p53 silencing partially restores anchorage-independent growth potential of the revertant through the suppression of anoikis. In addition, we identified a novel gene, named PHTS, with putative tumor suppressor properties, and showed that this gene is also involved in HeLaHF reversion independently of the p53 pathway. Expression profiling revealed that PHTS is one of the genes that is up-regulated in HeLaHF but not in HeLa. It encodes a putative protein with CD59-like domains. RNAi-mediated PHTS silencing resulted in the partial restoration of transformation (anchorage-independent growth) in HeLaHF cells, similar to that of p53 gene silencing, implying its tumor suppressor effect. However, the observed increased transformation potential by PHTS silencing appears to be due to an increased anchorage-independent proliferation rate rather than suppression of anoikis, unlike the effect of p53 silencing. p53 silencing did not affect PHTS gene expression, and vice versa, suggesting PHTS may function in a new and p53-independent tumor suppressor pathway. Furthermore, over-expression of PHTS in different cancer cell lines, in addition to HeLa, reduces cell growth likely via induced apoptosis, confirming the broad PHTS tumor suppressor properties

  6. Comparison of pressure between barium reduction and air reduction of the intussusception in children

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Kyung Hwan; Park, Sang Gyu; Park, Choong Ki; Yoon, Jong Sup [Hallym University College of Medicine, Seoul (Korea, Republic of)

    1989-08-15

    There are many method of treatment of the intussusception in children, including surgery, barium reduction, air reduction, and saline enema under ultrasonographic monitoring. Among them, barium reduction and air reduction have been used widely as nonsurgical method of treatment in radiologic department. During barium reduction, the bottle filled with barium solution must not be elevated over a 3 feet from the operating table. In air reduction, diagnostic pressure is about 60 mmHg and pressure during reduction usually is maintained between 90 mmHg and 130 mmHg. The authors have studied about pressure difference of barium solution by changing height of the bottle filled with barium solution, and have compared with pressure in air reduction. The results are as follows; 1. Pressure of 20 w/v % barium solution at the 60 cm height is 44.4 mmHg, and 69.6 mmHg at the 90 cm height. 2. Pressure of 40 w/v % barium solution at the 60 cm height is 51.4 mmHg, and 80.1 mmHg at the 90 cm height. 3. Pressure of 40 w/v % barium solution at the 90cm height is much lower than the pressure maintained during air reduction, and this difference in pressure may be one of the causes of low reduction rate in barium reduction then air reduction. 4. The pressure gradient per 10 cm height is about 8.45 mmHg in 20 w/v % barium solution, about 9.21 mmHg in 30 w/v % barium solution, and about 9.72 mmHg 40 w/v % barium solution. 5. Intraluminal pressure difference between the barium reduction and the air reduction is probably of the major causes of rapid diagnosis and high reduction rate in the air reduction.

  7. Comparison of pressure between barium reduction and air reduction of the intussusception in children

    International Nuclear Information System (INIS)

    Lee, Kyung Hwan; Park, Sang Gyu; Park, Choong Ki; Yoon, Jong Sup

    1989-01-01

    There are many method of treatment of the intussusception in children, including surgery, barium reduction, air reduction, and saline enema under ultrasonographic monitoring. Among them, barium reduction and air reduction have been used widely as nonsurgical method of treatment in radiologic department. During barium reduction, the bottle filled with barium solution must not be elevated over a 3 feet from the operating table. In air reduction, diagnostic pressure is about 60 mmHg and pressure during reduction usually is maintained between 90 mmHg and 130 mmHg. The authors have studied about pressure difference of barium solution by changing height of the bottle filled with barium solution, and have compared with pressure in air reduction. The results are as follows; 1. Pressure of 20 w/v % barium solution at the 60 cm height is 44.4 mmHg, and 69.6 mmHg at the 90 cm height. 2. Pressure of 40 w/v % barium solution at the 60 cm height is 51.4 mmHg, and 80.1 mmHg at the 90 cm height. 3. Pressure of 40 w/v % barium solution at the 90cm height is much lower than the pressure maintained during air reduction, and this difference in pressure may be one of the causes of low reduction rate in barium reduction then air reduction. 4. The pressure gradient per 10 cm height is about 8.45 mmHg in 20 w/v % barium solution, about 9.21 mmHg in 30 w/v % barium solution, and about 9.72 mmHg 40 w/v % barium solution. 5. Intraluminal pressure difference between the barium reduction and the air reduction is probably of the major causes of rapid diagnosis and high reduction rate in the air reduction

  8. Descriptive analysis of the verbal behavior of a therapist: a known-group validity analysis of the putative behavioral functions involved in clinical interaction.

    Science.gov (United States)

    Virues-Ortega, Javier; Montaño-Fidalgo, Montserrat; Froján-Parga, María Xesús; Calero-Elvira, Ana

    2011-12-01

    This study analyzes the interobserver agreement and hypothesis-based known-group validity of the Therapist's Verbal Behavior Category System (SISC-INTER). The SISC-INTER is a behavioral observation protocol comprised of a set of verbal categories representing putative behavioral functions of the in-session verbal behavior of a therapist (e.g., discriminative, reinforcing, punishing, and motivational operations). The complete therapeutic process of a clinical case of an individual with marital problems was recorded (10 sessions, 8 hours), and data were arranged in a temporal sequence using 10-min periods. Hypotheses based on the expected performance of the putative behavioral functions portrayed by the SISC-INTER codes across prevalent clinical activities (i.e., assessing, explaining, Socratic method, providing clinical guidance) were tested using autoregressive integrated moving average (ARIMA) models. Known-group validity analyses provided support to all hypotheses. The SISC-INTER may be a useful tool to describe therapist-client interaction in operant terms. The utility of reliable and valid protocols for the descriptive analysis of clinical practice in terms of verbal behavior is discussed. Copyright © 2011. Published by Elsevier Ltd.

  9. FUNCTIONAL GENOMICS IDENTIFIES TIS21-DEPENDENT MECHANISMS AND PUTATIVE CANCER DRUG TARGETS UNDERLYING MEDULLOBLASTOMA SHH-TYPE DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Giulia Gentile

    2016-11-01

    Full Text Available We have recently generated a novel medulloblastoma (MB mouse model with activation of the Shh pathway and lacking the MB suppressor Tis21 (Patched1+-Tis21KO.ts main phenotype is a defect of migration of the cerebellar granule precursor cells (GCPs. By genomic analysis of GCPs in vivo, we identified as drug target and major responsible of this defect the down-regulation of the promigratory chemokine Cxcl3. Consequently, the GCPs remain longer in the cerebellum proliferative area, and the MB frequency is enhanced. Here, we further analyzed the genes deregulated in a Tis21-dependent manner (Patched1+-is21 wild-type versus Ptch1+-Tis21 knockout, among which are a number of down-regulated tumor inhibitors and up-regulated tumor facilitators, focusing on pathways potentially involved in the tumorigenesis and on putative new drug targets.The data analysis using bioinformatic tools revealed: i a link between the Shh signaling and the Tis21-dependent impairment of the GCPs migration, through a Shh-dependent deregulation of the clathrin-mediated chemotaxis operating in the primary cilium through the Cxcl3-Cxcr2 axis; ii a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype the neural cell type involved in group 3 MB; iii the identification of a subset of putative drug targets for MB, involved, among the others, in the regulation of Hippo signaling and centrosome assembly. Finally, our findings define also the role of Tis21 in the regulation of gene expression, through epigenetic and RNA processing mechanisms, influencing the fate of the GCPs.

  10. Synthesis on accumulation of putative neurotransmitters by cultured neural crest cells

    International Nuclear Information System (INIS)

    Maxwell, G.D.; Sietz, P.D.; Rafford, C.E.

    1982-01-01

    The events mediating the differentiation of embryonic neural crest cells into several types of neurons are incompletely understood. In order to probe one aspect of this differentiation, we have examined the capacity of cultured quail trunk neural crest cells to synthesize, from radioactive precursors, and store several putative neurotransmitter compounds. These neural crest cultures develop the capacity to synthesize and accumulate acetylcholine and the catecholamines norepinephrine and dopamine. In contrast, detectable but relatively little synthesis and accumulation of 5-hydroxytryptamine gamma-aminobutyric acid, or octopamine from the appropriate radiolabeled precursors were observed. The capacity for synthesis and accumulation of radiolabeled acetylcholine and catecholamines is very low or absent at 2 days in vitro. Between 3 and 7 days in vitro, there is a marked rise in both catecholamine and acetylcholine accumulation in the cultures. These findings suggest that, under the particular conditions used in these experiments, the development of neurotransmitter biosynthesis in trunk neural crest cells ijs restricted and resembles, at least partially, the pattern observed in vivo. The development of this capacity to synthesize and store radiolabeled acetylcholine and catecholamines from the appropriate radioactive precursors coincides closely with the development of the activities of the synthetic enzymes choline acetyltransferase and dopamine beta-hydroxylase reported by others

  11. Differential proteomic analysis of Aspergillus fumigatus morphotypes reveals putative drug targets.

    Science.gov (United States)

    Kubitschek-Barreira, Paula H; Curty, Nathalia; Neves, Gabriela W P; Gil, Concha; Lopes-Bezerra, Leila M

    2013-01-14

    Aspergillus fumigatus is the main etiological agent of invasive aspergillosis, an important opportunistic infection for neutropenic patients. The main risk groups are patients with acute leukemia and bone marrow transplantation recipients. The lack of an early diagnostic test together with the limited spectrum of antifungal drugs remains a setback to the successful treatment of this disease. During invasive infection the inhaled fungal conidia enter the morphogenic cycle leading to angioinvasive hyphae. This work aimed to study differentially expressed proteins of A. fumigatus during morphogenesis. To achieve this goal, a 2D-DIGE approach was applied to study surface proteins extractable by reducing agents of two A. fumigatus morphotypes: germlings and hyphae. Sixty-three differentially expressed proteins were identified by MALDI-ToF/MS. We observed that proteins associated with biosynthetic pathways and proteins with multiple functions (miscellaneous) were over-expressed in the early stages of germination, while in hyphae, the most abundant proteins detected were related to metabolic processes or have unknown functions. Among the most interesting proteins regulated during morphogenesis, two putative drug targets were identified, the translational factor, eEF3 and the CipC-like protein. Neither of these proteins are present in mammalian cells. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. ESTs Analysis of Putative Genes Engaged in Polyporus umbellatus Sclerotial Development

    Directory of Open Access Journals (Sweden)

    Chao Song

    2014-09-01

    Full Text Available Polyporus umbellatus is one of the most widely used and precious medicinal fungi and the underground sclerotia are known to be with great medicinal value. However, the molecular mechanisms involved in sclerotial development are poorly understood. In the present study, we constructed a forward suppression subtractive hybridization (SSH cDNA library of Polyporus umbellatus to identify genes expressing differently between mycelium and sclerotia. In this library, a total of 1202 clones were sequenced, assembled into 222 contigs and 524 singletons which were further searched against the NCBI nonredundant (NR protein database (E-value cutoff, 10−5. Based on sequence similarity with known proteins, 378 sequences between mycelium and sclerotial were identified and classified into different functional categories through Gene Ontology (GO, Clusters of orthologous Groups of proteins (COGs. We have finally identified a majority of differentially expressed genes (constituting 5.6% of the present library between the two different periods. An expression level of 32 selected expressed sequence tags (ESTs generated from the above SSH cDNA library was studied through RT-PCR. This study provides the first global overview of genes putatively involved in Polyporus umbellatus sclerotial development and provides a preliminary basis for further functional research in terms of regulated gene expression in sclerotial production.

  13. Structure of a putative trans-editing enzyme for prolyl-tRNA synthetase from Aeropyrum pernix K1 at 1.7 Å resolution

    International Nuclear Information System (INIS)

    Murayama, Kazutaka; Kato-Murayama, Miyuki; Katsura, Kazushige; Uchikubo-Kamo, Tomomi; Yamaguchi-Hirafuji, Machiko; Kawazoe, Masahito; Akasaka, Ryogo; Hanawa-Suetsugu, Kyoko; Hori-Takemoto, Chie; Terada, Takaho; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2004-01-01

    The three-dimensional structure of the APE2540 protein from A. pernix K1 has been determined by the multiple anomalous dispersion method at 1.7 Å resolution. The structure includes two monomers in the asymmetric unit and shares structural similarity with the YbaK protein or cysteinyl-tRNA Pro deacylase from H. influenzae. The crystal structure of APE2540, the putative trans-editing enzyme ProX from Aeropyrum pernix K1, was determined in a high-throughput manner. The crystal belongs to the monoclinic space group P2 1 , with unit-cell parameters a = 47.4, b = 58.9, c = 53.6 Å, β = 106.8°. The structure was solved by the multiwavelength anomalous dispersion method at 1.7 Å and refined to an R factor of 16.8% (R free = 20.5%). The crystal structure includes two protein molecules in the asymmetric unit. Each monomer consists of eight β-strands and seven α-helices. A structure-homology search revealed similarity between the trans-editing enzyme YbaK (or cysteinyl-tRNA Pro deacylase) from Haemophilus influenzae (HI1434; 22% sequence identity) and putative ProX proteins from Caulobacter crescentus (16%) and Agrobacterium tumefaciens (21%)

  14. Detection of low frequency multi-drug resistance and novel putative maribavir resistance in immunocompromised paediatric patients with cytomegalovirus

    Directory of Open Access Journals (Sweden)

    Charlotte Jane Houldcroft

    2016-09-01

    Full Text Available Human cytomegalovirus (HCMV is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed paediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1-27 weeks. Changes in consensus sequence and resistance mutations were analysed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54 and C480F (UL97. In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of eleven subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome.

  15. Characterization of a putative cis-regulatory element that controls transcriptional activity of the pig uroplakin II gene promoter

    International Nuclear Information System (INIS)

    Kwon, Deug-Nam; Park, Mi-Ryung; Park, Jong-Yi; Cho, Ssang-Goo; Park, Chankyu; Oh, Jae-Wook; Song, Hyuk; Kim, Jae-Hwan; Kim, Jin-Hoi

    2011-01-01

    Highlights: → The sequences of -604 to -84 bp of the pUPII promoter contained the region of a putative negative cis-regulatory element. → The core promoter was located in the 5F-1. → Transcription factor HNF4 can directly bind in the pUPII core promoter region, which plays a critical role in controlling promoter activity. → These features of the pUPII promoter are fundamental to development of a target-specific vector. -- Abstract: Uroplakin II (UPII) is a one of the integral membrane proteins synthesized as a major differentiation product of mammalian urothelium. UPII gene expression is bladder specific and differentiation dependent, but little is known about its transcription response elements and molecular mechanism. To identify the cis-regulatory elements in the pig UPII (pUPII) gene promoter region, we constructed pUPII 5' upstream region deletion mutants and demonstrated that each of the deletion mutants participates in controlling the expression of the pUPII gene in human bladder carcinoma RT4 cells. We also identified a new core promoter region and putative negative cis-regulatory element within a minimal promoter region. In addition, we showed that hepatocyte nuclear factor 4 (HNF4) can directly bind in the pUPII core promoter (5F-1) region, which plays a critical role in controlling promoter activity. Transient cotransfection experiments showed that HNF4 positively regulates pUPII gene promoter activity. Thus, the binding element and its binding protein, HNF4 transcription factor, may be involved in the mechanism that specifically regulates pUPII gene transcription.

  16. Reduction - competitive tomorrow

    International Nuclear Information System (INIS)

    Worley, L.; Bargerstock, S.

    1995-01-01

    Inventory reduction is one of the few initiatives that represent significant cost-reduction potential that does not result in personnel reduction. Centerior Energy's Perry nuclear power plant has embarked on an aggressive program to reduce inventory while maintaining plant material availability. Material availability to the plant was above 98%, but at an unacceptable 1994 inventory book value of $47 million with inventory carrying costs calculated at 30% annually

  17. Breast Reduction Surgery

    Science.gov (United States)

    ... considering breast reduction surgery, consult a board-certified plastic surgeon. It's important to understand what breast reduction surgery entails — including possible risks and complications — as ...

  18. Cloning, expression, crystallization and preliminary X-ray analysis of a putative multiple antibiotic resistance repressor protein (MarR) from Xanthomonas campestris

    International Nuclear Information System (INIS)

    Tu, Zhi-Le; Li, Juo-Ning; Chin, Ko-Hsin; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Lyu, Ping-Chiang; Gao, Fei Philip; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-01-01

    A putative repressor for the multiple antibiotic resistance operon from a plant pathogen X. campestris pv. campestris has been overexpressed in E. coli, purified and crystallized. The crystals diffracted to 2.3 Å with good quality. The multiple antibiotic resistance operon (marRAB) is a member of the multidrug-resistance system. When induced, this operon enhances resistance of bacteria to a variety of medically important antibiotics, causing a serious global health problem. MarR is a marR-encoded protein that represses the transcription of the marRAB operon. Through binding with salicylate and certain antibiotics, however, MarR can derepress and activate the marRAB operon. In this report, the cloning, expression, crystallization and preliminary X-ray analysis of XC1739, a putative MarR repressor protein present in the Xanthomonas campestris pv. campestris, a Gram-negative bacterium causing major worldwide disease of cruciferous crops, are described. The XC1739 crystals diffracted to a resolution of at least 1.8 Å. They are orthorhombic and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 39.5, b = 54.2 and c = 139.5 Å, respectively. They contain two molecules in the asymmetric unit from calculation of the self-rotation function

  19. Whole-transcriptome survey of the putative ATP-binding cassette (ABC) transporter family genes in the latex-producing laticifers of Hevea brasiliensis.

    Science.gov (United States)

    Zhiyi, Nie; Guijuan, Kang; Yu, Li; Longjun, Dai; Rizhong, Zeng

    2015-01-01

    The ATP-binding cassette (ABC) proteins or transporters constitute a large protein family in plants and are involved in many different cellular functions and processes, including solute transportation, channel regulation and molecular switches, etc. Through transcriptome sequencing, a transcriptome-wide survey and expression analysis of the ABC protein genes were carried out using the laticiferous latex from Hevea brasiliensis (rubber tree). A total of 46 putative ABC family proteins were identified in the H. brasiliensis latex. These consisted of 12 'full-size', 21 'half-size' and 13 other putative ABC proteins, and all of them showed strong conservation with their Arabidopsis thaliana counterparts. This study indicated that all eight plant ABC protein paralog subfamilies were identified in the H. brasiliensis latex, of which ABCB, ABCG and ABCI were the most abundant. Real-time quantitative reverse transcription-polymerase chain reaction assays demonstrated that gene expression of several latex ABC proteins was regulated by ethylene, jasmonic acid or bark tapping (a wound stress) stimulation, and that HbABCB15, HbABCB19, HbABCD1 and HbABCG21 responded most significantly of all to the abiotic stresses. The identification and expression analysis of the latex ABC family proteins could facilitate further investigation into their physiological involvement in latex metabolism and rubber biosynthesis by H. brasiliensis.

  20. The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins

    Directory of Open Access Journals (Sweden)

    Cristian Oliver

    2017-09-01

    Full Text Available Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

  1. The Proteome of Biologically Active Membrane Vesicles from Piscirickettsia salmonis LF-89 Type Strain Identifies Plasmid-Encoded Putative Toxins.

    Science.gov (United States)

    Oliver, Cristian; Hernández, Mauricio A; Tandberg, Julia I; Valenzuela, Karla N; Lagos, Leidy X; Haro, Ronie E; Sánchez, Patricio; Ruiz, Pamela A; Sanhueza-Oyarzún, Constanza; Cortés, Marcos A; Villar, María T; Artigues, Antonio; Winther-Larsen, Hanne C; Avendaño-Herrera, Ruben; Yáñez, Alejandro J

    2017-01-01

    Piscirickettsia salmonis is the predominant bacterial pathogen affecting the Chilean salmonid industry. This bacterium is the etiological agent of piscirickettsiosis, a significant fish disease. Membrane vesicles (MVs) released by P. salmonis deliver several virulence factors to host cells. To improve on existing knowledge for the pathogenicity-associated functions of P. salmonis MVs, we studied the proteome of purified MVs from the P. salmonis LF-89 type strain using multidimensional protein identification technology. Initially, the cytotoxicity of different MV concentration purified from P. salmonis LF-89 was confirmed in an in vivo adult zebrafish infection model. The cumulative mortality of zebrafish injected with MVs showed a dose-dependent pattern. Analyses identified 452 proteins of different subcellular origins; most of them were associated with the cytoplasmic compartment and were mainly related to key functions for pathogen survival. Interestingly, previously unidentified putative virulence-related proteins were identified in P. salmonis MVs, such as outer membrane porin F and hemolysin. Additionally, five amino acid sequences corresponding to the Bordetella pertussis toxin subunit 1 and two amino acid sequences corresponding to the heat-labile enterotoxin alpha chain of Escherichia coli were located in the P. salmonis MV proteome. Curiously, these putative toxins were located in a plasmid region of P. salmonis LF-89. Based on the identified proteins, we propose that the protein composition of P. salmonis LF-89 MVs could reflect total protein characteristics of this P. salmonis type strain.

  2. Metallothermic reduction of molybdate

    International Nuclear Information System (INIS)

    Mukherjee, T.K.; Bose, D.K.

    1987-01-01

    This paper gives a brief account of the investigations conducted so far on metallothermic reduction of high grade molybdenite with particular emphasis on the work carried out in Bhabha Atomic Research Centre. Based on thermochemical considerations, the paper first introduces a number of metallic reductants suitable for use in metallothermic reduction of molybdenite. Aluminium, sodium and tin are found to be suitable reducing agents and very rightly they have found most applications in the research and development efforts on metallothermic reduction of molybdenite. The reduction with tin was conducted on fairly large scale both in vacuum and hydrogen atmosphere. The reaction was reported to be invariant depending mainly on the reduction temperature and a temperature of the order of 1250deg to 1300degC was required for good metal recovery. In comparison to tin, aluminothermic reduction of molybdenite was studied more extensively and it was conducted in closed bomb, vacuum and also in open atmosphere. In aluminothermic reduction, the influence of amount of reducing agent, amount of heat booster, preheating temperature and charging procedure on these metal yield was studied in detail. The reduction generally yielded massive molybdenum metal contaminated with aluminium as the major impurity element. Efforts were made to purify the reduced metal by arc melting, electron beam melting and molten salt electrorefining. 9 refs. (author)

  3. Original and regenerating lizard tail cartilage contain putative resident stem/progenitor cells.

    Science.gov (United States)

    Alibardi, Lorenzo

    2015-11-01

    Regeneration of cartilaginous tissues is limited in mammals but it occurs with variable extension in lizards (reptiles), including in their vertebrae. The ability of lizard vertebrae to regenerate cartilaginous tissue that is later replaced with bone has been analyzed using tritiated thymidine autoradiography and 5BrdU immunocytochemistry after single pulse or prolonged-pulse and chase experiments. The massive cartilage regeneration that can restore broad vertebral regions and gives rise to a long cartilaginous tube in the regenerating tail, depends from the permanence of some chondrogenic cells within adult vertebrae. Few cells that retain tritiated thymidine or 5-bromodeoxy-uridine for over 35 days are mainly localized in the inter-vertebral cartilage and in sparse chondrogenic regions of the neural arch of the vertebrae, suggesting that they are putative resident stem/progenitor cells. The study supports previous hypothesis indicating that the massive regeneration of the cartilaginous tissue in damaged vertebrae and in the regenerating tail of lizards derive from resident stem cells mainly present in the cartilaginous areas of the vertebrae including in the perichondrium that are retained in adult lizards as growing centers for most of their lifetime. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Antagonistic activity expressed by Shigella sonnei: identification of a putative new bacteriocin

    Directory of Open Access Journals (Sweden)

    Mireille Angela Bernardes Sousa

    2013-09-01

    Full Text Available Bacteriocins are antibacterial, proteinaceous substances that mediate microbial dynamics. Bacteriocin production is a highly disseminated property among all major lineages of bacteria, including Shigella. In this paper, we addressed the purification and characterisation of a bacteriocin produced by a Shigella sonnei strain (SS9 isolated from a child with acute diarrhoea. The substance was purified through ammonium-sulphate precipitation and sequential steps of chromatography. The intracellular fraction obtained at 75% ammonium sulphate maintained activity following exposure to pH values from 1-11 and storage at -80ºC for more than two years and was inactivated by high temperatures and proteases. The molecular mass of the purified bacteriocin was determined by mass spectrometry to be 18.56 kDa. The N-terminal sequence of the bacteriocin did not match any other antibacterial proteins described. A putative new bacteriocin produced by S. sonnei has been detected. This bacteriocin may represent a newly described protein or a previously described protein with a newly detected function. Considering that SS9 expresses antagonism against other diarrhoeagenic bacteria, the bacteriocin may contribute to S. sonnei virulence and is potentially applicable to either preventing or controlling diarrhoeal disease.

  5. Circulating miRNAs as Putative Biomarkers of Exercise Adaptation in Endurance Horses

    Directory of Open Access Journals (Sweden)

    Katia Cappelli

    2018-04-01

    Full Text Available Endurance exercise induces metabolic adaptations and has recently been reported associated with the modulation of a particular class of small noncoding RNAs, microRNAs, that act as post-transcriptional regulators of gene expression. Released into body fluids, they termed circulating miRNAs, and they have been recognized as more effective and accurate biomarkers than classical serum markers. This study examined serum profile of miRNAs through massive parallel sequencing in response to prolonged endurance exercise in samples obtained from four competitive Arabian horses before and 2 h after the end of competition. MicroRNA identification, differential gene expression (DGE analysis and a protein-protein interaction (PPI network showing significantly enriched pathways of target gene clusters, were assessed and explored. Our results show modulation of more than 100 miRNAs probably arising from tissues involved in exercise responses and indicating the modulation of correlated processes as muscle remodeling, immune and inflammatory responses. Circulating miRNA high-throughput sequencing is a promising approach for sports medicine for the discovery of putative biomarkers for predicting risks related to prolonged activity and monitoring metabolic adaptations.

  6. Exploring associations between gaze patterns and putative human mirror neuron system activity.

    Science.gov (United States)

    Donaldson, Peter H; Gurvich, Caroline; Fielding, Joanne; Enticott, Peter G

    2015-01-01

    The human mirror neuron system (MNS) is hypothesized to be crucial to social cognition. Given that key MNS-input regions such as the superior temporal sulcus are involved in biological motion processing, and mirror neuron activity in monkeys has been shown to vary with visual attention, aberrant MNS function may be partly attributable to atypical visual input. To examine the relationship between gaze pattern and interpersonal motor resonance (IMR; an index of putative MNS activity), healthy right-handed participants aged 18-40 (n = 26) viewed videos of transitive grasping actions or static hands, whilst the left primary motor cortex received transcranial magnetic stimulation. Motor-evoked potentials recorded in contralateral hand muscles were used to determine IMR. Participants also underwent eyetracking analysis to assess gaze patterns whilst viewing the same videos. No relationship was observed between predictive gaze and IMR. However, IMR was positively associated with fixation counts in areas of biological motion in the videos, and negatively associated with object areas. These findings are discussed with reference to visual influences on the MNS, and the possibility that MNS atypicalities might be influenced by visual processes such as aberrant gaze pattern.

  7. Exploring associations between gaze patterns and putative human mirror neuron system activity

    Directory of Open Access Journals (Sweden)

    Peter Hugh Donaldson

    2015-07-01

    Full Text Available The human mirror neuron system (MNS is hypothesised to be crucial to social cognition. Given that key MNS-input regions such as the superior temporal sulcus are involved in biological motion processing, and mirror neuron activity in monkeys has been shown to vary with visual attention, aberrant MNS function may be partly attributable to atypical visual input. To examine the relationship between gaze pattern and interpersonal motor resonance (IMR; an index of putative MNS activity, healthy right-handed participants aged 18-40 (n = 26 viewed videos of transitive grasping actions or static hands, whilst the left primary motor cortex received transcranial magnetic stimulation (TMS. Motor-evoked potentials (MEPs recorded in contralateral hand muscles were used to determine IMR. Participants also underwent eyetracking analysis to assess gaze patterns whilst viewing the same videos. No relationship was observed between predictive gaze (PG and IMR. However, IMR was positively associated with fixation counts in areas of biological motion in the videos, and negatively associated with object areas. These findings are discussed with reference to visual influences on the MNS, and the possibility that MNS atypicalities might be influenced by visual processes such as aberrant gaze pattern.

  8. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane.

    Science.gov (United States)

    Lahoda, Maryna; Chaloupkova, Radka; Stsiapanava, Alena; Damborsky, Jiri; Kuta Smatanova, Ivana

    2011-03-01

    Haloalkane dehalogenases hydrolyze carbon-halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure-function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP.

  9. Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis.

    Science.gov (United States)

    Stsiapanava, Alena; Koudelakova, Tana; Lapkouski, Mikalai; Pavlova, Martina; Damborsky, Jiri; Smatanova, Ivana Kuta

    2008-02-01

    The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P2(1)2(1)2(1), while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 A, respectively.

  10. Baclofen reverses the reduction in prepulse inhibition of the acoustic startle response induced by dizocilpine, but not by apomorphine.

    Science.gov (United States)

    Bortolato, Marco; Frau, Roberto; Aru, Gian Nicola; Orrù, Marco; Gessa, Gian Luigi

    2004-01-01

    Since baclofen, the prototypical GABA(B) receptor agonist, is known to reduce the activity of dopaminergic mesolimbic neurons, a putative antipsychotic property of this compound has been suggested, but the evidence for this is still controversial. The aim of the present study was to elucidate the effects of baclofen on the prepulse inhibition (PPI) of the acoustic startle response (ASR), a behavioral paradigm considered to be one of the most powerful tools for the evaluation of sensorimotor gating and for the screening of antipsychotics. We tested the effects of baclofen (1.25, 2.5, 5 and 10 mg/kg IP) in rats, per se and in co-treatment with some of the substances known to induce a robust reduction of PPI, such as apomorphine (0.25 mg/kg SC) and dizocilpine (0.1 mg/kg SC). Finally, in order to ascertain whether the effects of baclofen could be ascribed to its activity on GABA(B) receptors, we analyzed whether its action could be prevented by pretreatment with SCH 50911, a selective GABA(B) receptor antagonist (20 mg/kg IP). All the experiments were carried out using standard procedures for the assessment of PPI of the ASR. Baclofen per se produced no significant change in PPI parameters. Moreover, while no effect on apomorphine-mediated alterations in PPI parameters was observed, baclofen proved able to reverse dizocilpine-induced PPI disruption, and this effect was significantly prevented by SCH 50911. On the other hand, this last compound exhibited no effects per se at the same dose. These results indicate that GABA(B) receptors are implicated in the neurobiological circuitry accounting for glutamatergic action in sensorimotor gating, and therefore can be proposed as putative new targets in the pharmacological therapy of psychotic disorders. Further studies should be addressed to evaluate more closely the clinical efficacy of baclofen in this respect.

  11. A randomized trial of pneumatic reduction versus hydrostatic reduction for intussusception in pediatric patients.

    Science.gov (United States)

    Xie, Xiaolong; Wu, Yang; Wang, Qi; Zhao, Yiyang; Chen, Guobin; Xiang, Bo

    2017-08-08

    Data of randomly controlled trials comparing the hydrostatic and pneumatic reduction for intussusception in pediatric patients as initial therapy are lacking. The aim of this study was to conduct a randomly controlled trial to compare the effectiveness and safety of the hydrostatic and pneumatic reduction techniques. All intussusception patients who visited West China Hospital of Sichuan University from January 2014 to December 2015 were enrolled in this study in which they underwent pneumatic reduction or hydrostatic reduction. Patients were randomized into ultrasound-guided hydrostatic or X-ray-guided pneumatic reduction group. The data collected includes demographic data, symptoms, signs, and investigations. The primary outcome of the study was the success rate of reduction. And the secondary outcomes of the study were the rates of intestinal perforations and recurrence. A total of 124 children with intussusception who had met the inclusion criteria were enrolled. The overall success rate of this study was 90.32%. Univariable analysis showed that the success rate of hydrostatic reduction with normal saline (96.77%) was significantly higher than that of pneumatic reduction with air (83.87%) (p=0.015). Perforation after reduction was found in only one of the pneumatic reduction group. The recurrence rate of intussusception in the hydrostatic reduction group was 4.84% compared with 3.23% of pneumatic reduction group. Our study found that ultrasound-guided hydrostatic reduction is a simple, safe and effective nonoperative treatment for pediatric patients suffering from intussusceptions, and should be firstly adopted in the treatment of qualified patients. Therapeutic study TYPE OF STUDY: Prospective study. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Finding optimal exact reducts

    KAUST Repository

    AbouEisha, Hassan M.

    2014-01-01

    The problem of attribute reduction is an important problem related to feature selection and knowledge discovery. The problem of finding reducts with minimum cardinality is NP-hard. This paper suggests a new algorithm for finding exact reducts

  13. Sequence analysis of the MYC oncogene involved in the t(8;14)(q24;q11) chromosome translocation in a human leukemia T-cell line indicates that putative regulatory regions are not altered

    International Nuclear Information System (INIS)

    Finver, S.N.; Nishikura, K.; Finger, L.R.; Haluska, F.G.; Finan, J.; Nowell, P.C.; Croce, C.M.

    1988-01-01

    The authors cloned the translocation-associated and homologous normal MYC alleles from SKW-3, a leukemia T-cell line with the t(8; 14)(q24; q11) translocation, and determined the sequence of the MYC oncogene first exon and flanking 5' putative regulatory regions. S1 nuclease protection experiments utilizing a MYC first exon probe demonstrated transcriptional deregulation of the MYC gene associated with the T-cell receptor α locus on the 8q + chromosome of SKW-3 cells. Nucleotide sequence analysis of the translocation-associated (8q +) MYC allele identified a single base substitution within the upstream flanking region; the homologous nontranslocated allele contained an additional substitution and a two-base deletion. None of the deletions or substitutions localized to putative 5' regulatory regions. The MYC first exon sequence was germ line in both alleles. These results demonstrate that alterations within the putative 5' MYC regulatory regions are not necessarily involved in MYC deregulation in T-cell leukemias, and they show that juxtaposition of the T-cell receptor α locus to a germ-line MYC oncogene results in MYC deregulation

  14. Putative adaptive inter-slope divergence of transposon frequency in fruit flies (Drosophila melanogaster) at "Evolution Canyon", Mount Carmel, Israel.

    Science.gov (United States)

    Beiles, Avigdor; Raz, Shmuel; Ben-Abu, Yuval; Nevo, Eviatar

    2015-10-14

    The current analysis of transposon elements (TE) in Drosophila melanogaster at Evolution Canyon, (EC), Israel, is based on data and analysis done by our collaborators (Drs. J. Gonzalez, J. Martinez and W. Makalowski, this issue). They estimated the frequencies of 28 TEs (transposon elements) in fruit flies (D. melanogaster) from the ecologically tropic, hot, and dry south-facing slope (SFS) or "African" slope (AS) of EC and compared it with the TE frequencies on the temperate-cool and humid north-facing slope (NFS) or "European" slope (ES), separated, on average, by 250 m. The flies were sampled from two stations on each slope. We received their results, including the frequencies of each TE on each slope, and the probabilities of the statistical analyses (G-tests) of each TE separately. We continued the analysis of the inter-slope differences of the frequencies of the TEs, and based our different conclusions on that analysis and on the difference between micro (=EC) and macro (2000 km.) comparisons [Gonzalez et al. 2015 doi: 10.1186/s13062-015-0075-4 ]. Our collaborators based all their conclusions on the non-significant results of each of the individual tests of the 28 TEs. We analysed also the distribution of the TE differences between the slopes, based on their results. Thirteen TEs were more frequent on the SFS, 11 were more frequent on the NFS, and four had equal frequencies. Because of the equalizing effect of the ongoing migration, only small and temporary differences between the slopes (0 - 0.06) were regarded by us as random fluctuations (drift). Three TEs were intermediate (0.08-0.09) and await additional research. The 11 TEs with large frequency differences (0.12 - 0.22) were regarded by us as putative adaptive TEs, because the equalizing power of ongoing migration will eliminate random large differences. Five of them were higher on the SFS and six were higher on the NFS. Gaps in the distribution of the differences distinguished between the large and

  15. Identification of putative QTLs for seedling stage phosphorus starvation response in finger millet (Eleusine coracana L. Gaertn. by association mapping and cross species synteny analysis.

    Directory of Open Access Journals (Sweden)

    M Ramakrishnan

    Full Text Available A germplasm assembly of 128 finger millet genotypes from 18 countries was evaluated for seedling-stage phosphorus (P responses by growing them in P sufficient (Psuf and P deficient (Pdef treatments. Majority of the genotypes showed adaptive responses to low P condition. Based on phenotype behaviour using the best linear unbiased predictors for each trait, genotypes were classified into, P responsive, low P tolerant and P non-responsive types. Based on the overall phenotype performance under Pdef, 10 genotypes were identified as low P tolerants. The low P tolerant genotypes were characterised by increased shoot and root length and increased root hair induction with longer root hairs under Pdef, than under Psuf. Association mapping of P response traits using mixed linear models revealed four quantitative trait loci (QTLs. Two QTLs (qLRDW.1 and qLRDW.2 for low P response affecting root dry weight explained over 10% phenotypic variation. In silico synteny analysis across grass genomes for these QTLs identified putative candidate genes such as Ser-Thr kinase and transcription factors such as WRKY and basic helix-loop-helix (bHLH. The QTLs for response under Psuf were mapped for traits such as shoot dry weight (qHSDW.1 and root length (qHRL.1. Putative associations of these QTLs over the syntenous regions on the grass genomes revealed proximity to cytochrome P450, phosphate transporter and pectin methylesterase inhibitor (PMEI genes. This is the first report of the extent of phenotypic variability for P response in finger millet genotypes during seedling-stage, along with the QTLs and putative candidate genes associated with P starvation tolerance.

  16. Identification of putative QTLs for seedling stage phosphorus starvation response in finger millet (Eleusine coracana L. Gaertn.) by association mapping and cross species synteny analysis.

    Science.gov (United States)

    Ramakrishnan, M; Ceasar, S Antony; Vinod, K K; Duraipandiyan, V; Ajeesh Krishna, T P; Upadhyaya, Hari D; Al-Dhabi, N A; Ignacimuthu, S

    2017-01-01

    A germplasm assembly of 128 finger millet genotypes from 18 countries was evaluated for seedling-stage phosphorus (P) responses by growing them in P sufficient (Psuf) and P deficient (Pdef) treatments. Majority of the genotypes showed adaptive responses to low P condition. Based on phenotype behaviour using the best linear unbiased predictors for each trait, genotypes were classified into, P responsive, low P tolerant and P non-responsive types. Based on the overall phenotype performance under Pdef, 10 genotypes were identified as low P tolerants. The low P tolerant genotypes were characterised by increased shoot and root length and increased root hair induction with longer root hairs under Pdef, than under Psuf. Association mapping of P response traits using mixed linear models revealed four quantitative trait loci (QTLs). Two QTLs (qLRDW.1 and qLRDW.2) for low P response affecting root dry weight explained over 10% phenotypic variation. In silico synteny analysis across grass genomes for these QTLs identified putative candidate genes such as Ser-Thr kinase and transcription factors such as WRKY and basic helix-loop-helix (bHLH). The QTLs for response under Psuf were mapped for traits such as shoot dry weight (qHSDW.1) and root length (qHRL.1). Putative associations of these QTLs over the syntenous regions on the grass genomes revealed proximity to cytochrome P450, phosphate transporter and pectin methylesterase inhibitor (PMEI) genes. This is the first report of the extent of phenotypic variability for P response in finger millet genotypes during seedling-stage, along with the QTLs and putative candidate genes associated with P starvation tolerance.

  17. Exome sequencing in 53 sporadic cases of schizophrenia identifies 18 putative candidate genes.

    Directory of Open Access Journals (Sweden)

    Michel Guipponi

    Full Text Available Schizophrenia (SCZ is a severe, debilitating mental illness which has a significant genetic component. The identification of genetic factors related to SCZ has been challenging and these factors remain largely unknown. To evaluate the contribution of de novo variants (DNVs to SCZ, we sequenced the exomes of 53 individuals with sporadic SCZ and of their non-affected parents. We identified 49 DNVs, 18 of which were predicted to alter gene function, including 13 damaging missense mutations, 2 conserved splice site mutations, 2 nonsense mutations, and 1 frameshift deletion. The average number of exonic DNV per proband was 0.88, which corresponds to an exonic point mutation rate of 1.7×10(-8 per nucleotide per generation. The non-synonymous-to-synonymous mutation ratio of 2.06 did not differ from neutral expectations. Overall, this study provides a list of 18 putative candidate genes for sporadic SCZ, and when combined with the results of similar reports, identifies a second proband carrying a non-synonymous DNV in the RGS12 gene.

  18. Putative Stem Cells in Human Dental Pulp with Irreversible Pulpitis-An Exploratory Study

    Science.gov (United States)

    Wang, Z.; Pan, J.; Wright, JT; Bencharit, S.; Zhang, S.; Everett, ET; Teixeira, FB; Preisser, JS

    2010-01-01

    Introduction Although human dental pulp stem cells isolated from healthy teeth have been extensively characterized, it is unknown whether stem cells also exist in clinically compromised teeth with irreversible pulpitis. Here we explored whether cells retrieved from clinically compromised dental pulp have stem cell-like properties. Methods Pulp cells were isolated from healthy teeth (control group) and from teeth with clinically diagnosed irreversible pulpitis (diseased group). Cell proliferation, stem cell marker STRO-1 expression and cell odonto-osteo-genic differentiation competence were compared. Results Cells from the diseased group demonstrated decreased colony formation capacity and a slightly decreased cell proliferation rate but had similar STRO-1 expression, and exhibited a similar percentage of positive ex vivo osteogenic induction and dentin sialophosphoprotein expression from STRO-1-enriched pulp cells. Conclusion Our study provides preliminary evidence that clinically compromised dental pulp may contain putative cells with certain stem cell properties. Further characterization of these cells will provide insight regarding whether they could serve as a source of endogenous multipotent cells in tissue regeneration based dental pulp therapy. PMID:20416426

  19. A Sequence and Structure Based Method to Predict Putative Substrates, Functions and Regulatory Networks of Endo Proteases

    Science.gov (United States)

    Venkatraman, Prasanna; Balakrishnan, Satish; Rao, Shashidhar; Hooda, Yogesh; Pol, Suyog

    2009-01-01

    Background Proteases play a central role in cellular homeostasis and are responsible for the spatio- temporal regulation of function. Many putative proteases have been recently identified through genomic approaches, leading to a surge in global profiling attempts to characterize their function. Through such efforts and others it has become evident that many proteases play non-traditional roles. Accordingly, the number and the variety of the substrate repertoire of proteases are expected to be much larger than previously assumed. In line with such global profiling attempts, we present here a method for the prediction of natural substrates of endo proteases (human proteases used as an example) by employing short peptide sequences as specificity determinants. Methodology/Principal Findings Our method incorporates specificity determinants unique to individual enzymes and physiologically relevant dual filters namely, solvent accessible surface area-a parameter dependent on protein three-dimensional structure and subcellular localization. By incorporating such hitherto unused principles in prediction methods, a novel ligand docking strategy to mimic substrate binding at the active site of the enzyme, and GO functions, we identify and perform subjective validation on putative substrates of matriptase and highlight new functions of the enzyme. Using relative solvent accessibility to rank order we show how new protease regulatory networks and enzyme cascades can be created. Conclusion We believe that our physiologically relevant computational approach would be a very useful complementary method in the current day attempts to profile proteases (endo proteases in particular) and their substrates. In addition, by using functional annotations, we have demonstrated how normal and unknown functions of a protease can be envisaged. We have developed a network which can be integrated to create a proteolytic world. This network can in turn be extended to integrate other regulatory

  20. Reduction of U3O8 to U by a metallic reductant, Li

    International Nuclear Information System (INIS)

    Jin-Mok Hur; Sun-Seok Hong; Hansoo Lee

    2010-01-01

    Reduction of U 3 O 8 was investigated for the recycling of spent oxide fuel from a commercial nuclear power plant. The possible reduction methods were proposed and compared. Based on the thermodynamic analysis, Li metal was selected as a reductant. The optimum reaction temperature for the reduction of U 3 O 8 was investigated at the wider reaction temperature range. The adverse oxidation of U metal by Li 2 O at 1,000 deg C was experimentally verified. Ellingham diagram was constructed to investigate the extent of the uranium oxides reduction when the reaction was carried out above melting point of U metal. (author)

  1. Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane

    Czech Academy of Sciences Publication Activity Database

    Lahoda, M.; Chaloupková, R.; Stsiapanava, A.; Damborský, J.; Kutá-Smatanová, Ivana

    2011-01-01

    Roč. 67, Part 3 (2011), s. 397-400 ISSN 1744-3091 R&D Projects: GA MŠk(CZ) LC06010; GA MŠk(CZ) ED1.1.00/02.0073 Institutional research plan: CEZ:AV0Z60870520 Keywords : haloalkane dehalogenases * DhaA * Rhodococcus rhodochrous Subject RIV: CE - Biochemistry Impact factor: 0.506, year: 2011

  2. Elucidating the Molecular Basis and Regulation of Chromium(VI) Reduction by Shewanella oneidensis MR-1 and Resistance to Metal Toxicity Using Integrated Biochemical, Genomic and Proteomic Approaches

    Energy Technology Data Exchange (ETDEWEB)

    Dorothea K. Thompson; Robert Hettich

    2007-02-06

    Shewanella oneidensis MR-1 is a model environmental organism that possesses diverse respiratory capacities, including the ability to reduce soluble Cr(VI) to sparingly soluble, less toxic Cr(III). Chromate is a serious anthropogenic pollutant found in subsurface sediment and groundwater environments due to its widespread use in defense and industrial applications. Effective bioremediation of chromate-contaminated sites requires knowledge of the molecular mechanisms and regulation of heavy metal resistance and biotransformation by dissimilatory metal-reducing bacteria. Towards this goal, our ERSP-funded work was focused on the identification and functional analysis of genes/proteins comprising the response pathways for chromate detoxification and/or reduction. Our work utilized temporal transcriptomic profiling and whole-cell proteomic analyses to characterize the dynamic molecular response of MR-1 to an acute chromate shock (up to 90 min) as well as to a 24-h, low-dose exposure. In addition, we have examined the transcriptome of MR-1 cells actively engaged in chromate reduction. These studies implicated the involvement of a functionally undefined DNA-binding response regulator (SO2426) and a putative azoreductase (SO3585) in the chromate stress response of MR-1.

  3. Isolation and Expression analysis of OsPME1, encoding for a putative Pectin Methyl Esterase from Oryza sativa (subsp. indica)

    OpenAIRE

    Kanneganti, Vydehi; Gupta, Aditya Kumar

    2009-01-01

    Pectin Methyl Esterases (PMEs) play an essential role during plant development by affecting the mechanical properties of the plant cell walls. Recent studies indicated that PMEs play important role in pollen tube development. In this study, we isolated a 1.3 kb cDNA clone from rice panicle cDNA library. It contained a 1038 bp of open reading frame (ORF) encoding for a putative pectin methyl esterase of 345 aminoacids with a 20 aminoacid signal peptide and was hence designated as OsPME1 (Oryza...

  4. Effect of the deletion of qmoABC and the promoter distal gene encoding a hypothetical protein on sulfate-reduction in Desulfovibrio vulgaris Hildenborough

    Energy Technology Data Exchange (ETDEWEB)

    Zane, Grant M.; Yen, Huei-chi Bill; Wall, Judy D.

    2010-03-18

    The pathway of electrons required for the reduction of sulfate in sulfate-reducing bacteria (SRB) is not yet fully characterized. In order to determine the role of a transmembrane protein complex suggested to be involved in this process, a deletion of Desulfovibrio vulgaris Hildenborough was created by marker exchange mutagenesis that eliminated four genes putatively encoding the QmoABC complex and a hypothetical protein (DVU0851). The Qmo complex (quinone-interacting membrane-bound oxidoreductase) is proposed to be responsible for transporting electrons to the dissimilatory adenosine-5?phosphosulfate (APS) reductase in SRB. In support of the predicted role of this complex, the deletion mutant was unable to grow using sulfate as its sole electron acceptor with a range of electron donors. To explore a possible role for the hypothetical protein in sulfate reduction, a second mutant was constructed that had lost only the gene that codes for DVU0851. The second constructed mutant grew with sulfate as the sole electron acceptor; however, there was a lag that was not present with the wild-type or complemented strain. Neither deletion strain was significantly impaired for growth with sulfite or thiosulfate as terminal electron acceptor. Complementation of the D(qmoABC-DVU0851) mutant with all four genes or only the qmoABC genes restored its ability to grow by sulfate respiration. These results confirmed the prediction that the Qmo complex is in the electron pathway for sulfate-reduction and revealed that no other transmembrane complex could compensate when Qmo was lacking.

  5. HaploReg v4: systematic mining of putative causal variants, cell types, regulators and target genes for human complex traits and disease.

    Science.gov (United States)

    Ward, Lucas D; Kellis, Manolis

    2016-01-04

    More than 90% of common variants associated with complex traits do not affect proteins directly, but instead the circuits that control gene expression. This has increased the urgency of understanding the regulatory genome as a key component for translating genetic results into mechanistic insights and ultimately therapeutics. To address this challenge, we developed HaploReg (http://compbio.mit.edu/HaploReg) to aid the functional dissection of genome-wide association study (GWAS) results, the prediction of putative causal variants in haplotype blocks, the prediction of likely cell types of action, and the prediction of candidate target genes by systematic mining of comparative, epigenomic and regulatory annotations. Since first launching the website in 2011, we have greatly expanded HaploReg, increasing the number of chromatin state maps to 127 reference epigenomes from ENCODE 2012 and Roadmap Epigenomics, incorporating regulator binding data, expanding regulatory motif disruption annotations, and integrating expression quantitative trait locus (eQTL) variants and their tissue-specific target genes from GTEx, Geuvadis, and other recent studies. We present these updates as HaploReg v4, and illustrate a use case of HaploReg for attention deficit hyperactivity disorder (ADHD)-associated SNPs with putative brain regulatory mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  6. Communities of Putative Ericoid Mycorrhizal Fungi Isolated from Alpine Dwarf Shrubs in Japan: Effects of Host Identity and Microhabitat.

    Science.gov (United States)

    Koizumi, Takahiko; Nara, Kazuhide

    2017-06-24

    Dwarf shrubs of the family Ericaceae are common in arctic and alpine regions. Many of these plants are associated with ericoid mycorrhizal (ERM) fungi, which allow them to take nutrients and water from the soil under harsh environmental conditions and, thus, affect host plant survival. Despite the importance of ERM fungi to alpine plant communities, limited information is available on the effects of microhabitat and host identity on ERM fungal communities. We investigated the communities of putative ERM fungi isolated from five dwarf shrub species (Arcterica nana, Diapensia lapponica, Empetrum nigrum, Loiseleuria procumbens, and Vaccinium vitis-idaea) that co-occur in an alpine region of Japan, with reference to distinct microhabitats provided by large stone pine (Pinus pumila) shrubs (i.e. bare ground, the edge of stone pine shrubs, and the inside of stone pine shrubs). We obtained 703 fungal isolates from 222 individual plants. These isolates were classified into 55 operational taxonomic units (OTUs) based on the sequencing of internal transcribed spacer regions in ribosomal DNA. These putative ERM fungal communities were dominated by Helotiales fungi for all host species. Cistella and Trimmatostroma species, which have rarely been detected in ERM roots in previous studies, were abundant. ERM fungal communities were significantly different among microhabitats (R 2 =0.28), while the host effect explained less variance in the fungal communities after excluding the microhabitat effect (R 2 =0.17). Our results suggest that the host effect on ERM fungal communities is minor and the distributions of hosts and fungal communities may be assessed based on microhabitat conditions.

  7. Identification of putative regulatory motifs in the upstream regions of co-expressed functional groups of genes in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Joshi NV

    2009-01-01

    Full Text Available Abstract Background Regulation of gene expression in Plasmodium falciparum (Pf remains poorly understood. While over half the genes are estimated to be regulated at the transcriptional level, few regulatory motifs and transcription regulators have been found. Results The study seeks to identify putative regulatory motifs in the upstream regions of 13 functional groups of genes expressed in the intraerythrocytic developmental cycle of Pf. Three motif-discovery programs were used for the purpose, and motifs were searched for only on the gene coding strand. Four motifs – the 'G-rich', the 'C-rich', the 'TGTG' and the 'CACA' motifs – were identified, and zero to all four of these occur in the 13 sets of upstream regions. The 'CACA motif' was absent in functional groups expressed during the ring to early trophozoite transition. For functional groups expressed in each transition, the motifs tended to be similar. Upstream motifs in some functional groups showed 'positional conservation' by occurring at similar positions relative to the translational start site (TLS; this increases their significance as regulatory motifs. In the ribonucleotide synthesis, mitochondrial, proteasome and organellar translation machinery genes, G-rich, C-rich, CACA and TGTG motifs, respectively, occur with striking positional conservation. In the organellar translation machinery group, G-rich motifs occur close to the TLS. The same motifs were sometimes identified for multiple functional groups; differences in location and abundance of the motifs appear to ensure different modes of action. Conclusion The identification of positionally conserved over-represented upstream motifs throws light on putative regulatory elements for transcription in Pf.

  8. Reduction operators of Burgers equation.

    Science.gov (United States)

    Pocheketa, Oleksandr A; Popovych, Roman O

    2013-02-01

    The solution of the problem on reduction operators and nonclassical reductions of the Burgers equation is systematically treated and completed. A new proof of the theorem on the special "no-go" case of regular reduction operators is presented, and the representation of the coefficients of operators in terms of solutions of the initial equation is constructed for this case. All possible nonclassical reductions of the Burgers equation to single ordinary differential equations are exhaustively described. Any Lie reduction of the Burgers equation proves to be equivalent via the Hopf-Cole transformation to a parameterized family of Lie reductions of the linear heat equation.

  9. Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

    Science.gov (United States)

    Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

    2016-01-01

    Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.

  10. The effects of promising to tell the truth, the putative confession, and recall and recognition questions on maltreated and non-maltreated children's disclosure of a minor transgression.

    Science.gov (United States)

    Quas, Jodi A; Stolzenberg, Stacia N; Lyon, Thomas D

    2018-02-01

    This study examined the utility of two interview instructions designed to overcome children's reluctance to disclose transgressions: eliciting a promise from children to tell the truth and the putative confession (telling children that a suspect "told me everything that happened and wants you to tell the truth"). The key questions were whether the instructions increased disclosure in response to recall questions and in response to recognition questions that were less or more explicit about transgressions and whether instructions were differentially effective with age. A total sample of 217 4- to 9-year-old maltreated and comparable non-maltreated children and a stranger played with a set of toys. For half of the children within each group, two of the toys appeared to break while they were playing. The stranger admonished secrecy. Shortly thereafter, children were questioned about what happened in one of three interview conditions. Some children were asked to promise to tell the truth. Others were given the putative confession, and still others received no interview instructions. When coupled with recall questions, the promise was effective at increasing disclosures only among older children, whereas the putative confession was effective regardless of age. Across interview instruction conditions, recognition questions that did not suggest wrongdoing elicited few additional transgression disclosures, whereas recognition questions that explicitly mentioned wrongdoing elicited some true reports but also some false alarms. No differences in disclosure emerged between maltreated and non-maltreated children. Results highlight the potential benefits and limitations of different interviewing approaches when questioning reluctant children. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Active3 noise reduction

    International Nuclear Information System (INIS)

    Holzfuss, J.

    1996-01-01

    Noise reduction is a problem being encountered in a variety of applications, such as environmental noise cancellation, signal recovery and separation. Passive noise reduction is done with the help of absorbers. Active noise reduction includes the transmission of phase inverted signals for the cancellation. This paper is about a threefold active approach to noise reduction. It includes the separation of a combined source, which consists of both a noise and a signal part. With the help of interaction with the source by scanning it and recording its response, modeling as a nonlinear dynamical system is achieved. The analysis includes phase space analysis and global radial basis functions as tools for the prediction used in a subsequent cancellation procedure. Examples are given which include noise reduction of speech. copyright 1996 American Institute of Physics

  12. Vietnam; Poverty Reduction Strategy Paper

    OpenAIRE

    International Monetary Fund

    2004-01-01

    This paper assesses the Poverty Reduction Strategy Paper of Vietnam, known as the Comprehensive Poverty Reduction and Growth Strategy (CPRGS). It is an action program to achieve economic growth and poverty reduction objectives. This paper reviews the objectives and tasks of socio-economic development and poverty reduction. The government of Vietnam takes poverty reduction as a cutting-through objective in the process of country socio-economic development and declares its commitment to impleme...

  13. Genome sequence and comparative analysis of a putative entomopathogenic Serratia isolated from Caenorhabditis briggsae.

    Science.gov (United States)

    Abebe-Akele, Feseha; Tisa, Louis S; Cooper, Vaughn S; Hatcher, Philip J; Abebe, Eyualem; Thomas, W Kelley

    2015-07-18

    Entomopathogenic associations between nematodes in the genera Steinernema and Heterorhabdus with their cognate bacteria from the bacterial genera Xenorhabdus and Photorhabdus, respectively, are extensively studied for their potential as biological control agents against invasive insect species. These two highly coevolved associations were results of convergent evolution. Given the natural abundance of bacteria, nematodes and insects, it is surprising that only these two associations with no intermediate forms are widely studied in the entomopathogenic context. Discovering analogous systems involving novel bacterial and nematode species would shed light on the evolutionary processes involved in the transition from free living organisms to obligatory partners in entomopathogenicity. We report the complete genome sequence of a new member of the enterobacterial genus Serratia that forms a putative entomopathogenic complex with Caenorhabditis briggsae. Analysis of the 5.04 MB chromosomal genome predicts 4599 protein coding genes, seven sets of ribosomal RNA genes, 84 tRNA genes and a 64.8 KB plasmid encoding 74 genes. Comparative genomic analysis with three of the previously sequenced Serratia species, S. marcescens DB11 and S. proteamaculans 568, and Serratia sp. AS12, revealed that these four representatives of the genus share a core set of ~3100 genes and extensive structural conservation. The newly identified species shares a more recent common ancestor with S. marcescens with 99% sequence identity in rDNA sequence and orthology across 85.6% of predicted genes. Of the 39 genes/operons implicated in the virulence, symbiosis, recolonization, immune evasion and bioconversion, 21 (53.8%) were present in Serratia while 33 (84.6%) and 35 (89%) were present in Xenorhabdus and Photorhabdus EPN bacteria respectively. The majority of unique sequences in Serratia sp. SCBI (South African Caenorhabditis briggsae Isolate) are found in ~29 genomic islands of 5 to 65 genes and are

  14. MCNP variance reduction overview

    International Nuclear Information System (INIS)

    Hendricks, J.S.; Booth, T.E.

    1985-01-01

    The MCNP code is rich in variance reduction features. Standard variance reduction methods found in most Monte Carlo codes are available as well as a number of methods unique to MCNP. We discuss the variance reduction features presently in MCNP as well as new ones under study for possible inclusion in future versions of the code

  15. Putative bacterial interactions from metagenomic knowledge with an integrative systems ecology approach.

    Science.gov (United States)

    Bordron, Philippe; Latorre, Mauricio; Cortés, Maria-Paz; González, Mauricio; Thiele, Sven; Siegel, Anne; Maass, Alejandro; Eveillard, Damien

    2016-02-01

    Following the trend of studies that investigate microbial ecosystems using different metagenomic techniques, we propose a new integrative systems ecology approach that aims to decipher functional roles within a consortium through the integration of genomic and metabolic knowledge at genome scale. For the sake of application, using public genomes of five bacterial strains involved in copper bioleaching: Acidiphilium cryptum, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans, we first reconstructed a global metabolic network. Next, using a parsimony assumption, we deciphered sets of genes, called Sets from Genome Segments (SGS), that (1) are close on their respective genomes, (2) take an active part in metabolic pathways and (3) whose associated metabolic reactions are also closely connected within metabolic networks. Overall, this SGS paradigm depicts genomic functional units that emphasize respective roles of bacterial strains to catalyze metabolic pathways and environmental processes. Our analysis suggested that only few functional metabolic genes are horizontally transferred within the consortium and that no single bacterial strain can accomplish by itself the whole copper bioleaching. The use of SGS pinpoints a functional compartmentalization among the investigated species and exhibits putative bacterial interactions necessary for promoting these pathways. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  16. Differential protein accumulations in isolates of the strawberry wilt pathogen Fusarium oxysporum f. sp. fragariae differing in virulence.

    Science.gov (United States)

    Fang, Xiangling; Barbetti, Martin J

    2014-08-28

    This study was conducted to define differences in Fusarium oxysporum f. sp. fragariae (Fof) isolates with different virulence efficiency to strawberry at the proteome level, in combination with their differences in mycelial growth, conidial production and germination. Comparative proteome analyses revealed substantial differences in mycelial proteomes between Fof isolates, where the 54 differentially accumulated protein spots were consistently over-accumulated or exclusively in the highly virulent isolate. These protein spots were identified through MALDI-TOF/TOF mass spectrometry analyses, and the identified proteins were mainly related to primary and protein metabolism, antioxidation, electron transport, cell cycle and transcription based on their putative functions. Proteins of great potential as Fof virulence factors were those involved in ubiquitin/proteasome-mediated protein degradation and reactive oxygen species detoxification; the hydrolysis-related protein haloacid dehalogenase superfamily hydrolase; 3,4-dihydroxy-2-butanone 4-phosphate synthase associated with riboflavin biosynthesis; and those exclusive to the highly virulent isolate. In addition, post-translational modifications may also make an important contribution to Fof virulence. F. oxysporum f. sp. fragariae (Fof), the causal agent of Fusarium wilt in strawberry, is a serious threat to commercial strawberry production worldwide. However, factors and mechanisms contributing to Fof virulence remained unknown. This study provides knowledge of the molecular basis for the differential expression of virulence in Fof, allowing new possibilities towards developing alternative and more effective strategies to manage Fusarium wilt. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Identification of putative agouti-related protein(87-132)-melanocortin-4 receptor interactions by homology molecular modeling and validation using chimeric peptide ligands.

    Science.gov (United States)

    Wilczynski, Andrzej; Wang, Xiang S; Joseph, Christine G; Xiang, Zhimin; Bauzo, Rayna M; Scott, Joseph W; Sorensen, Nicholas B; Shaw, Amanda M; Millard, William J; Richards, Nigel G; Haskell-Luevano, Carrie

    2004-04-22

    Agouti-related protein (AGRP) is one of only two naturally known antagonists of G-protein-coupled receptors (GPCRs) identified to date. Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis. Alpha-melanocyte stimulating hormone (alpha-MSH) is one of the known endogenous agonists for these melanocortin receptors. Insight into putative interactions between the antagonist AGRP amino acids with the melanocortin-4 receptor (MC4R) may be important for the design of unique ligands for the treatment of obesity related diseases and is currently lacking in the literature. A three-dimensional homology molecular model of the mouse MC4 receptor complex with the hAGRP(87-132) ligand docked into the receptor has been developed to identify putative antagonist ligand-receptor interactions. Key putative AGRP-MC4R interactions include the Arg111 of hAGRP(87-132) interacting in a negatively charged pocket located in a cavity formed by transmembrane spanning (TM) helices 1, 2, 3, and 7, capped by the acidic first extracellular loop (EL1) and specifically with the conserved melanocortin receptor residues mMC4R Glu92 (TM2), mMC4R Asp114 (TM3), and mMC4R Asp118 (TM3). Additionally, Phe112 and Phe113 of hAGRP(87-132) putatively interact with an aromatic hydrophobic pocket formed by the mMC4 receptor residues Phe176 (TM4), Phe193 (TM5), Phe253 (TM6), and Phe254 (TM6). To validate the AGRP-mMC4R model complex presented herein from a ligand perspective, we generated nine chimeric peptide ligands based on a modified antagonist template of the hAGRP(109-118) (Tyr-c[Asp-Arg-Phe-Phe-Asn-Ala-Phe-Dpr]-Tyr-NH(2)). In these chimeric ligands, the antagonist AGRP Arg-Phe-Phe residues were replaced by the melanocortin agonist His/D-Phe-Arg-Trp amino acids. These peptides resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3-5Rs). The most notable results include the identification of a novel subnanomolar melanocortin peptide

  18. Redox cycles of vitamin E: Hydrolysis and ascorbic acid dependent reduction of 8a-(alkyldioxy)tocopherones

    International Nuclear Information System (INIS)

    Liebler, D.C.; Kaysen, K.L.; Kennedy, T.A.

    1989-01-01

    Oxidation of the biological antioxidant α-tocopherol (vitamin E; TH) by peroxyl radicals yields 8a-(alkyldioxy)tocopherones, which either may hydrolyze to α-tocopheryl quinone (TQ) or may be reduced by ascorbic acid to regenerate TH. To define the chemistry of this putative two-electron TH redox cycle, we studied the hydrolysis and reduction of 8a-[(2,4-dimethyl-1-nitrilopent-2-yl)dioxyl]tocopherone (1) in acetonitrile/buffer mixtures and in phospholipid liposomes. TQ formation in acetonitrile/buffer mixtures, which was monitored spectrophotometrically, declined with increasing pH and could not be detected above pH 4. The rate of TQ formation from 1 first increased with time and then decreased in a first-order terminal phase. Rearrangement of 8a-hydroxy-α-tocopherone (2) to TQ displayed first-order kinetics identical with the terminal phase for TQ formation from 1. Both rate constants increased with decreasing pH. Hydrolysis of 1 in acetonitrile/H 2 18 O yielded [ 18 O]TQ. These observations suggest that 1 loses the 8a-(alkyldioxy) moiety to produce the tocopherone cation (T + ), which hydrolyzes to 2, the TQ-forming intermediate. Incubation of either 1 or 2 with ascorbic acid in acetonitrile/buffer yielded TH. Reduction of both 1 and 2 decreased with increasing pH. In phosphatidylcholine liposomes at pH 7, approximately 10% of the T + generated from 1 was reduced to TH by 5 mM ascorbic acid. The results collectively demonstrate that T + is the ascorbic acid reducible intermediate in a two-electron TH redox cycle, a process that probably would require biocatalysis to proceed in biological membranes

  19. Reduction of blue tungsten oxide

    International Nuclear Information System (INIS)

    Wilken, T.; Wert, C.; Woodhouse, J.; Morcom, W.

    1975-01-01

    A significant portion of commercial tungsten is produced by hydrogen reduction of oxides. Although several modes of reduction are possible, hydrogen reduction is used where high purity tungsten is required and where the addition of other elements or compounds is desired for modification of the metal, as is done for filaments in the lamp industry. Although several investigations of the reduction of oxides have been reported (1 to 5), few principles have been developed which can aid in assessment of current commercial practice. The reduction process was examined under conditions approximating commercial practice. The specific objectives were to determine the effects of dopants, of water vapor in the reducing atmosphere, and of reduction temperature upon: (1) the rate of the reaction by which blue tungsten oxide is reduced to tungsten metal, (2) the intermediate oxides associated with reduction, and (3) the morphology of the resulting tungsten powder

  20. Chromosomal locations of three human nuclear genes (RPSM12, TUFM, and AFG3L1) specifying putative components of the mitochondrial gene expression apparatus.

    Science.gov (United States)

    Shah, Z H; Migliosi, V; Miller, S C; Wang, A; Friedman, T B; Jacobs, H T

    1998-03-15

    We have mapped the chromosomal locations of three human nuclear genes for putative components of the apparatus of mitochondrial gene expression, using a combination of in situ hybridization and interspecies hybrid mapping. The genes RPMS12 (mitoribosomal protein S12, a conserved protein component of the mitoribosomal accuracy center), TUFM (mitochondrial elongation factor EF-Tu), and AFG3L1 (similar to the yeast genes Afg3 and Rca1 involved in the turnover of mistranslated or misfolded mtDNA-encoded polypeptides) were initially characterized by a combination of database sequence analysis, PCR, cloning, and DNA sequencing. RPMS12 maps to chromosome 19q13.1, close to the previously mapped gene for autosomal dominant hearing loss DFNA4. The TUFM gene is located on chromosome 16p11.2, with a putative pseudogene or variant (TUFML) located very close to the centromere of chromosome 17. AFG3L1 is located on chromosome 16q24, very close to the telomere. By virtue of their inferred functions in mitochondria, these genes should be regarded as candidates of disorders sharing features with mitochondrial disease syndromes, such as sensorineural deafness, diabetes, and retinopathy.

  1. Finding optimal exact reducts

    KAUST Repository

    AbouEisha, Hassan M.

    2014-01-01

    The problem of attribute reduction is an important problem related to feature selection and knowledge discovery. The problem of finding reducts with minimum cardinality is NP-hard. This paper suggests a new algorithm for finding exact reducts with minimum cardinality. This algorithm transforms the initial table to a decision table of a special kind, apply a set of simplification steps to this table, and use a dynamic programming algorithm to finish the construction of an optimal reduct. I present results of computer experiments for a collection of decision tables from UCIML Repository. For many of the experimented tables, the simplification steps solved the problem.

  2. Molecular cloning of a novel glucuronokinase/putative pyrophosphorylase from zebrafish acting in an UDP-glucuronic acid salvage pathway.

    Directory of Open Access Journals (Sweden)

    Roman Gangl

    Full Text Available In animals, the main precursor for glycosaminoglycan and furthermore proteoglycan biosynthesis, like hyaluronic acid, is UDP-glucuronic acid, which is synthesized via the nucleotide sugar oxidation pathway. Mutations in this pathway cause severe developmental defects (deficiency in the initiation of heart valve formation. In plants, UDP-glucuronic acid is synthesized via two independent pathways. Beside the nucleotide sugar oxidation pathway, a second minor route to UDP-glucuronic acid exist termed the myo-inositol oxygenation pathway. Within this myo-inositol is ring cleaved into glucuronic acid, which is subsequently converted to UDP-glucuronic acid by glucuronokinase and UDP-sugar pyrophosphorylase. Here we report on a similar, but bifunctional enzyme from zebrafish (Danio rerio which has glucuronokinase/putative pyrophosphorylase activity. The enzyme can convert glucuronic acid into UDP-glucuronic acid, required for completion of the alternative pathway to UDP-glucuronic acid via myo-inositol and thus establishes a so far unknown second route to UDP-glucuronic acid in animals. Glucuronokinase from zebrafish is a member of the GHMP-kinase superfamily having unique substrate specificity for glucuronic acid with a Km of 31 ± 8 µM and accepting ATP as the only phosphate donor (Km: 59 ± 9 µM. UDP-glucuronic acid pyrophosphorylase from zebrafish has homology to bacterial nucleotidyltransferases and requires UTP as nucleosid diphosphate donor. Genes for bifunctional glucuronokinase and putative UDP-glucuronic acid pyrophosphorylase are conserved among some groups of lower animals, including fishes, frogs, tunicates, and polychaeta, but are absent from mammals. The existence of a second pathway for UDP-glucuronic acid biosynthesis in zebrafish likely explains some previous contradictory finding in jekyll/ugdh zebrafish developmental mutants, which showed residual glycosaminoglycans and proteoglycans in knockout mutants of UDP

  3. INDUSTRIAL BOILER RETROFIT FOR NOX CONTROL: COMBINED SELECTIVE NONCATALYTIC REDUCTION AND SELECTIVE CATALYTIC REDUCTION

    Science.gov (United States)

    The paper describes retrofitting and testing a 590 kW (2 MBtu/hr), oil-fired, three-pass, fire-tube package boiler with a combined selective noncatalytic reduction (SNCR) and selective catalytic reduction (SCR) system. The system demonstrated 85% nitrogen oxides (NOx) reduction w...

  4. Occurrence of Putative Virulence Genes in Arcobacter Species Isolated from Humans and Animals

    Science.gov (United States)

    Douidah, Laid; de Zutter, Lieven; Baré, Julie; De Vos, Paul; Vandamme, Peter; Vandenberg, Olivier; Van den Abeele, Anne-Marie

    2012-01-01

    Interest in arcobacters in veterinary and human public health has increased since the first report of the isolation of arcobacters from food of animal origin. Since then, studies worldwide have reported the occurrence of arcobacters on food and in food production animals and have highlighted possible transmission, especially of Arcobacter butzleri, to the human population. In humans, arcobacters are associated with enteritis and septicemia. To assess their clinical relevance for humans and animals, evaluation of potential virulence factors is required. However, up to now, little has been known about the mechanisms of pathogenicity. Because of their close phylogenetic affiliation to the food-borne pathogen Campylobacter and their similar clinical manifestations, the presence of nine putative Campylobacter virulence genes (cadF, ciaB, cj1349, hecA, hecB, irgA, mviN, pldA, and tlyA) previously identified in the recent Arcobacter butzleri ATCC 49616 genome sequence was determined in a large set of human and animal Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii strains after the development of rapid and accurate PCR assays and confirmed by sequencing and dot blot hybridization. PMID:22170914

  5. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes.

    Science.gov (United States)

    Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2016-01-01

    Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy.

  6. Comparative Transcriptome Analysis Identifies Putative Genes Involved in Steroid Biosynthesis in Euphorbia tirucalli

    Directory of Open Access Journals (Sweden)

    Weibo Qiao

    2018-01-01

    Full Text Available Phytochemical analysis of different Euphorbia tirucalli tissues revealed a contrasting tissue-specificity for the biosynthesis of euphol and β-sitosterol, which represent the two pharmaceutically active steroids in E. tirucalli. To uncover the molecular mechanism underlying this tissue-specificity for phytochemicals, a comprehensive E. tirucalli transcriptome derived from its root, stem, leaf and latex was constructed, and a total of 91,619 unigenes were generated with 51.08% being successfully annotated against the non-redundant (Nr protein database. A comparison of the transcriptome from different tissues discovered members of unigenes in the upstream steps of sterol backbone biosynthesis leading to this tissue-specific sterol biosynthesis. Among them, the putative oxidosqualene cyclase (OSC encoding genes involved in euphol synthesis were notably identified, and their expressions were significantly up-regulated in the latex. In addition, genome-wide differentially expressed genes (DEGs in the different E. tirucalli tissues were identified. The cluster analysis of those DEGs showed a unique expression pattern in the latex compared with other tissues. The DEGs identified in this study would enrich the insights of sterol biosynthesis and the regulation mechanism of this latex-specificity.

  7. Dolochar as a reductant in the reduction roasting of iron ore slimes

    Science.gov (United States)

    Rath, Swagat S.; Rao, Danda Srinivas

    2017-12-01

    The present investigation examines the viability of dolochar, a sponge iron industry waste material, as a reductant in the reduction roasting of iron ore slimes, which are another waste generated by iron ore beneficiation plants. Under statistically determined optimum conditions, which include a temperature of 900°C, a reductant-to-feed mass ratio of 0.35, and a reduction time of 30-45 min, the roasted mass, after being subjected to low-intensity magnetic separation, yielded an iron ore concentrate of approximately 64wt% Fe at a mass recovery of approximately 71% from the feed iron ore slime assaying 56.2wt% Fe. X-ray diffraction analyses indicated that the magnetic products contain magnetite and hematite as the major phases, whereas the nonmagnetic fractions contain quartz and hematite.

  8. Synchrotron- and focal plane array-based Fourier-transform infrared spectroscopy differentiates the basalis and functionalis epithelial endometrial regions and identifies putative stem cell regions of human endometrial glands.

    Science.gov (United States)

    Theophilou, Georgios; Morais, Camilo L M; Halliwell, Diane E; Lima, Kássio M G; Drury, Josephine; Martin-Hirsch, Pierre L; Stringfellow, Helen F; Hapangama, Dharani K; Martin, Francis L

    2018-05-09

    The cyclical process of regeneration of the endometrium suggests that it may contain a cell population that can provide daughter cells with high proliferative potential. These cell lineages are clinically significant as they may represent clonogenic cells that may also be involved in tumourigenesis as well as endometriotic lesion development. To determine whether the putative stem cell location within human uterine tissue can be derived using vibrational spectroscopy techniques, normal endometrial tissue was interrogated by two spectroscopic techniques. Paraffin-embedded uterine tissues containing endometrial glands were sectioned to 10-μm-thick parallel tissue sections and were floated onto BaF 2 slides for synchrotron radiation-based Fourier-transform infrared (SR-FTIR) microspectroscopy and globar focal plane array-based FTIR spectroscopy. Different spectral characteristics were identified depending on the location of the glands examined. The resulting infrared spectra were subjected to multivariate analysis to determine associated biophysical differences along the length of longitudinal and crosscut gland sections. Comparison of the epithelial cellular layer of transverse gland sections revealed alterations indicating the presence of putative transient-amplifying-like cells in the basalis and mitotic cells in the functionalis. SR-FTIR microspectroscopy of the base of the endometrial glands identified the location where putative stem cells may reside at the same time pointing towards ν s PO 2 - in DNA and RNA, nucleic acids and amide I and II vibrations as major discriminating factors. This study supports the view that vibration spectroscopy technologies are a powerful adjunct to our understanding of the stem cell biology of endometrial tissue. Graphical abstract ᅟ.

  9. Interaction mediated by the putative tip regions of MdsA and MdsC in the formation of a Salmonella-specific tripartite efflux pump.

    Directory of Open Access Journals (Sweden)

    Saemee Song

    Full Text Available To survive in the presence of a wide range of toxic compounds, gram-negative bacteria expel such compounds via tripartite efflux pumps that span both the inner and outer membranes. The Salmonella-specific MdsAB pump consists of MdsB, a resistance-nodulation-division (RND-type inner membrane transporter (IMT that requires the membrane fusion protein (MFP MdsA, and an outer membrane protein (OMP; MdsC or TolC to form a tripartite efflux complex. In this study, we investigated the role of the putative tip regions of MdsA and its OMPs, MdsC and TolC, in the formation of a functional MdsAB-mediated efflux pump. Comparative analysis indicated that although sequence homologies of MdsA and MdsC with other MFPs and OMPs, respectively, are extremely low, key residues in the putative tip regions of these proteins are well conserved. Mutagenesis studies on these conserved sites demonstrated their importance for the physical and functional interactions required to form an MdsAB-mediated pump. Our studies suggest that, despite differences in the primary amino acid sequences and functions of various OMPs and MFPs, interactions mediated by the conserved tip regions of OMP and MFP are required for the formation of functional tripartite efflux pumps in gram-negative bacteria.

  10. Supradural inflammatory soup in awake and freely moving rats induces facial allodynia that is blocked by putative immune modulators.

    Science.gov (United States)

    Wieseler, Julie; Ellis, Amanda; McFadden, Andrew; Stone, Kendra; Brown, Kimberley; Cady, Sara; Bastos, Leandro F; Sprunger, David; Rezvani, Niloofar; Johnson, Kirk; Rice, Kenner C; Maier, Steven F; Watkins, Linda R

    2017-06-01

    Facial allodynia is a migraine symptom that is generally considered to represent a pivotal point in migraine progression. Treatment before development of facial allodynia tends to be more successful than treatment afterwards. As such, understanding the underlying mechanisms of facial allodynia may lead to a better understanding of the mechanisms underlying migraine. Migraine facial allodynia is modeled by applying inflammatory soup (histamine, bradykinin, serotonin, prostaglandin E2) over the dura. Whether glial and/or immune activation contributes to such pain is unknown. Here we tested if trigeminal nucleus caudalis (Sp5C) glial and/or immune cells are activated following supradural inflammatory soup, and if putative glial/immune inhibitors suppress the consequent facial allodynia. Inflammatory soup was administered via bilateral indwelling supradural catheters in freely moving rats, inducing robust and reliable facial allodynia. Gene expression for microglial/macrophage activation markers, interleukin-1β, and tumor necrosis factor-α increased following inflammatory soup along with robust expression of facial allodynia. This provided the basis for pursuing studies of the behavioral effects of 3 diverse immunomodulatory drugs on facial allodynia. Pretreatment with either of two compounds broadly used as putative glial/immune inhibitors (minocycline, ibudilast) prevented the development of facial allodynia, as did treatment after supradural inflammatory soup but prior to the expression of facial allodynia. Lastly, the toll-like receptor 4 (TLR4) antagonist (+)-naltrexone likewise blocked development of facial allodynia after supradural inflammatory soup. Taken together, these exploratory data support that activated glia and/or immune cells may drive the development of facial allodynia in response to supradural inflammatory soup in unanesthetized male rats. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Buoyancy differences among two deepwater ciscoes from the Great Lakes and their putative ancestor

    Science.gov (United States)

    Krause, A.E.; Eshenroder, R.L.; Begnoche, L.J.

    2002-01-01

    We analyzed buoyancy in two deepwater ciscoes, Coregonus hoyi and C. kiyi, and in C. artedi, their putative ancestor, and also analyzed how variations in fish weight, water content, and lipid content affected buoyancy. Buoyancy was significantly different among the three species (p < 0.0001). Estimates of percent buoyancy (neutral buoyancy = 0.0%) were: kiyi, 3.8%; hoyi, 4.7%; and artedi, 5.7%. Buoyancy did not change with fish weight alone (p = 0.38). Fish weight was negatively related to water content for all three species (p = 0.037). Lipid content was not significantly different between hoyi and kiyi, but artedi had significantly fewer lipids than hoyi and kiyi (p < 0.10). When artedi was removed from the analysis, fish weight and lipids accounted for 48% of the variation in buoyancy (p = 0.003), fatter hoyi were less dense than leaner hoyi, but fatter and leaner kiyi were no different in density. Our findings provide additional evidence that buoyancy regulation was a speciating mechanism in deepwater ciscoes and that kiyi is more specialized than hoyi for diel-vertical migration in deep water.

  12. Motivation and personality: relationships between putative motive dimensions and the five factor model of personality.

    Science.gov (United States)

    Bernard, Larry C

    2010-04-01

    There are few multidimensional measures of individual differences in motivation available. The Assessment of Individual Motives-Questionnaire assesses 15 putative dimensions of motivation. The dimensions are based on evolutionary theory and preliminary evidence suggests the motive scales have good psychometric properties. The scales are reliable and there is evidence of their consensual validity (convergence of self-other ratings) and behavioral validity (relationships with self-other reported behaviors of social importance). Additional validity research is necessary, however, especially with respect to current models of personality. The present study tested two general and 24 specific hypotheses based on proposed evolutionary advantages/disadvantages and fitness benefits/costs of the five-factor model of personality together with the new motive scales in a sample of 424 participants (M age=28.8 yr., SD=14.6). Results were largely supportive of the hypotheses. These results support the validity of new motive dimensions and increase understanding of the five-factor model of personality.

  13. Antiviral Activities and Putative Identification of Compounds in Microbial Extracts from the Hawaiian Coastal Waters

    Directory of Open Access Journals (Sweden)

    Yuanan Lu

    2012-02-01

    Full Text Available Marine environments are a rich source of significant bioactive compounds. The Hawaiian archipelago, located in the middle of the Pacific Ocean, hosts diverse microorganisms, including many endemic species. Thirty-eight microbial extracts from Hawaiian coastal waters were evaluated for their antiviral activity against four mammalian viruses including herpes simplex virus type one (HSV-1, vesicular stomatitis virus (VSV, vaccinia virus and poliovirus type one (poliovirus-1 using in vitro cell culture assay. Nine of the 38 microbial crude extracts showed antiviral potencies and three of these nine microbial extracts exhibited significant activity against the enveloped viruses. A secosteroid, 5α(H,17α(H,(20R-beta-acetoxyergost-8(14-ene was putatively identified and confirmed to be the active compound in these marine microbial extracts. These results warrant future in-depth tests on the isolation of these active elements in order to explore and validate their antiviral potential as important therapeutic remedies.

  14. The anaerobic degradation of organic matter in Danish coastal sediments: iron reduction, manganese reduction, and sulfate reduction

    DEFF Research Database (Denmark)

    Canfield, Donald Eugene; Thamdrup, B; Hansen, Jens Würgler

    1993-01-01

    ). In the deep portion of the basin, surface Mn enrichments reached 3.5 wt%, and Mn reduction was the only important anaerobic carbon oxidation process in the upper 10 cm of the sediment. In the less Mn-rich sediments from intermediate depths in the basin, Fe reduction ranged from somewhat less, to far more...... speculate that in shallow sediments of the Skagerrak, surface Mn oxides are present in a somewhat reduced oxidation level (deep basin....

  15. An efficient cDNA-AFLP-based strategy for the identification of putative pathogenicity factors from the potato cyst nematode Globodera rostochiensis.

    Science.gov (United States)

    Qin, L; Overmars, H; Helder, J; Popeijus, H; van der Voort, J R; Groenink, W; van Koert, P; Schots, A; Bakker, J; Smant, G

    2000-08-01

    A new strategy has been designed to identify putative pathogenicity factors from the dorsal or subventral esophageal glands of the potato cyst nematode Globodera rostochiensis. Three independent criteria were used for selection. First, genes of interest should predominantly be expressed in infective second-stage juveniles, and not, or to a far lesser extent, in younger developmental stages. For this, gene expression profiles from five different developmental stages were generated with cDNA-AFLP (amplified fragment length polymorphism). Secondly, the mRNA corresponding to such a putative pathogenicity factor should predominantly be present in the esophageal glands of pre-parasitic juveniles. This was checked by in situ hybridization. As a third criterion, these proteinaceous factors should be preceded by a signal peptide for secretion. Expression profiles of more than 4,000 genes were generated and three up-regulated, dorsal gland-specific proteins preceded by signal peptide for secretion were identified. No dorsal gland genes have been cloned before from plant-parasitic nematodes. The partial sequence of these three factors, A4, A18, and A41, showed no significant homology to any known gene. Their presence in the dorsal glands of infective juveniles suggests that these proteins could be involved in feeding cell initiation, and not in migration in the plant root or in protection against plant defense responses. Finally, the applicability of this new strategy in other plant-microbe interactions is discussed.

  16. Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh [Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC (United States); Zhu, Hao [The Rutgers Center for Computational and Integrative Biology, Rutgers University, Camden, NJ (United States); Department of Chemistry, Rutgers University, Camden, NJ (United States); Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia [NovaMechanics Ltd., Nicosia (Cyprus); Rusyn, Ivan, E-mail: iir@unc.edu [Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC (United States); Tropsha, Alexander, E-mail: alex_tropsha@unc.edu [Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC (United States)

    2013-10-01

    Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function.

  17. Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

    International Nuclear Information System (INIS)

    Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh; Zhu, Hao; Afantitis, Antreas; Mouchlis, Varnavas D.; Melagraki, Georgia; Rusyn, Ivan; Tropsha, Alexander

    2013-01-01

    Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R 2 = 0.71, STL R 2 = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R 2 = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function. • The results

  18. An S/T-Q cluster domain census unveils new putative targets under Tel1/Mec1 control

    Directory of Open Access Journals (Sweden)

    Cheung Hannah C

    2012-11-01

    Full Text Available Abstract Background The cellular response to DNA damage is immediate and highly coordinated in order to maintain genome integrity and proper cell division. During the DNA damage response (DDR, the sensor kinases Tel1 and Mec1 in Saccharomyces cerevisiae and ATM and ATR in human, phosphorylate multiple mediators which activate effector proteins to initiate cell cycle checkpoints and DNA repair. A subset of kinase substrates are recognized by the S/T-Q cluster domain (SCD, which contains motifs of serine (S or threonine (T followed by a glutamine (Q. However, the full repertoire of proteins and pathways controlled by Tel1 and Mec1 is unknown. Results To identify all putative SCD-containing proteins, we analyzed the distribution of S/T-Q motifs within verified Tel1/Mec1 targets and arrived at a unifying SCD definition of at least 3 S/T-Q within a stretch of 50 residues. This new SCD definition was used in a custom bioinformatics pipeline to generate a census of SCD-containing proteins in both yeast and human. In yeast, 436 proteins were identified, a significantly larger number of hits than were expected by chance. These SCD-containing proteins did not distribute equally across GO-ontology terms, but were significantly enriched for those involved in processes related to the DDR. We also found a significant enrichment of proteins involved in telophase and cytokinesis, protein transport and endocytosis suggesting possible novel Tel1/Mec1 targets in these pathways. In the human proteome, a wide range of similar proteins were identified, including homologs of some SCD-containing proteins found in yeast. This list also included high concentrations of proteins in the Mediator, spindle pole body/centrosome and actin cytoskeleton complexes. Conclusions Using a bioinformatic approach, we have generated a census of SCD-containing proteins that are involved not only in known DDR pathways but several other pathways under Tel1/Mec1 control suggesting new

  19. Identification and characterization of putative stem cells in the adult pig ovary.

    Science.gov (United States)

    Bui, Hong-Thuy; Van Thuan, Nguyen; Kwon, Deug-Nam; Choi, Yun-Jung; Kang, Min-Hee; Han, Jae-Woong; Kim, Teoan; Kim, Jin-Hoi

    2014-06-01

    Recently, the concept of 'neo-oogenesis' has received increasing attention, since it was shown that adult mammals have a renewable source of eggs. The purpose of this study was to elucidate the origin of these eggs and to confirm whether neo-oogenesis continues throughout life in the ovaries of the adult mammal. Adult female pigs were utilized to isolate, identify and characterize, including their proliferation and differentiation capabilities, putative stem cells (PSCs) from the ovary. PSCs were found to comprise a heterogeneous population based on c-kit expression and cell size, and also express stem and germ cell markers. Analysis of PSC molecular progression during establishment showed that these cells undergo cytoplasmic-to-nuclear translocation of Oct4 in a manner reminiscent of gonadal primordial germ cells (PGCs). Hence, cells with the characteristics of early PGCs are present or are generated in the adult pig ovary. Furthermore, the in vitro establishment of porcine PSCs required the presence of ovarian cell-derived extracellular regulatory factors, which are also likely to direct stem cell niche interactions in vivo. In conclusion, the present work supports a crucial role for c-kit and kit ligand/stem cell factor in stimulating the growth, proliferation and nuclear reprogramming of porcine PSCs, and further suggests that porcine PSCs might be the culture equivalent of early PGCs. © 2014. Published by The Company of Biologists Ltd.

  20. Genetic Causes of Putative Autosomal Recessive Intellectual Disability Cases in Hamedan Province

    Directory of Open Access Journals (Sweden)

    Milad Bastami

    2012-04-01

    Full Text Available Objective: The aim of this study was to investigate the genetic causes of autosomal recessive intellectual disabilities (AR-ID in Hamadan province of Iran. Materials & Methods: In this descriptive-analytical cross-sectional study, 25 families with more than one affected with putative autosomal recessive intellectual disability were chosen with collaboration of Welfare Organization of Hamadan province. Families were included a total of 60 patients (39 male and 21 female whose intellectual disability had been confirmed by Raven IQ test. Each family was asked for clinical examination and getting consent form. Blood sample was collected from each family. One proband from each family was tested for CGG repeat expansion in FMR1 gene, chromosomal abnormalities and inborn errors of metabolism. We also performed homozygosity mapping based on STR markers for seven known MCPH loci in families with primary microcephaly and AR-ID. Results: Five families had full mutation of Fragile X syndrome. No chromosomal abnormalities were identified. Metabolic screening revealed one family with Medium Chain Acyl CoA Dehydrogenase deficiency. None of three families with primary microcephaly and AR-ID showed linkage to any of known seven MCPH loci. Conclusion: The main causes of ID in Hamadan province were Fragile X syndrome and Autosomal Recessive Primary Microcephaly with the frequencies of 20% and 12%, respectively.

  1. Air Layer Drag Reduction

    Science.gov (United States)

    Ceccio, Steven; Elbing, Brian; Winkel, Eric; Dowling, David; Perlin, Marc

    2008-11-01

    A set of experiments have been conducted at the US Navy's Large Cavitation Channel to investigate skin-friction drag reduction with the injection of air into a high Reynolds number turbulent boundary layer. Testing was performed on a 12.9 m long flat-plate test model with the surface hydraulically smooth and fully rough at downstream-distance-based Reynolds numbers to 220 million and at speeds to 20 m/s. Local skin-friction, near-wall bulk void fraction, and near-wall bubble imaging were monitored along the length of the model. The instrument suite was used to access the requirements necessary to achieve air layer drag reduction (ALDR). Injection of air over a wide range of air fluxes showed that three drag reduction regimes exist when injecting air; (1) bubble drag reduction that has poor downstream persistence, (2) a transitional regime with a steep rise in drag reduction, and (3) ALDR regime where the drag reduction plateaus at 90% ± 10% over the entire model length with large void fractions in the near-wall region. These investigations revealed several requirements for ALDR including; sufficient volumetric air fluxes that increase approximately with the square of the free-stream speed, slightly higher air fluxes are needed when the surface tension is reduced, higher air fluxes are required for rough surfaces, and the formation of ALDR is sensitive to the inlet condition.

  2. Comparative transcriptome analysis links distinct peritoneal tumor spread types, miliary and non-miliary, with putative origin, tubes and ovaries, in high grade serous ovarian cancer.

    Science.gov (United States)

    Auer, Katharina; Bachmayr-Heyda, Anna; Aust, Stefanie; Grunt, Thomas W; Pils, Dietmar

    2017-03-01

    High grade serous ovarian cancer (HGSOC) is characterized by extensive local, i.e. peritoneal, tumor spread, manifested in two different clinical presentations, miliary (many millet sized peritoneal implants) and non-miliary (few large exophytically growing peritoneal nodes), and an overall unfavorable outcome. HGSOC is thought to arise from fallopian tube secretory epithelial cells, via so called serous tubal intraepithelial carcinomas (STICs) but an ovarian origin was never ruled out for at least some cases. Comparative transcriptome analyses of isolated tumor cells from fresh HGSOC tissues and (immortalized) ovarian surface epithelial and fallopian tube secretory epithelial cell lines revealed a close relation between putative origin and tumor spread characteristic, i.e. miliary from tubes and non-miliary from ovaries. The latter were characterized by more mesenchymal cell characteristics, more adaptive tumor immune infiltration, and a favorable overall survival. Several molecular sub-classification systems (Crijns' overall survival signature, Yoshihara's subclasses, and a collagen-remodeling signature) seem to already indicate origin. Putative origin alone is a significant independent predictor for HGSOC outcome, validated in independent patient cohorts. Characteristics of both spread types could guide development of new targeted therapeutics, which are urgently needed. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Characterization of Chloroethylene Dehalogenation by Cell Extracts of Desulfomonile tiedjei and Its Relationship to Chlorobenzoate Dehalogenation

    Science.gov (United States)

    Townsend, G. T.; Suflita, J. M.

    1996-01-01

    We characterized the reductive dehalogenation of tetrachloroethylene in cell extracts of Desulfomonile tiedjei and compared it with this organism's 3-chlorobenzoate dehalogenation activity. Tetrachloroethylene was sequentially dehalogenated to trichloro- and dichloroethylene; there was no evidence for dichloroethylene dehalogenation. Like the previously characterized 3-chlorobenzoate dehalogenation activity, tetrachloroethylene dehalogenation was heat sensitive, not oxygen labile, and increased in proportion to the amount of protein in assay mixtures. In addition, both dehalogenation activities were dependent on hydrogen or formate as an electron donor and had an absolute requirement for either methyl viologen or triquat as an electron carrier in vitro. Both activities appear to be catalyzed by integral membrane proteins with similar solubilization characteristics. Dehalogenation of tetrachloroethylene was inhibited by 3-chlorobenzoate but not by the structural isomers 2- and 4-chlorobenzoate. The last two compounds are not substrates for D. tiedjei. These findings lead us to suggest that the dehalogenation of tetrachloroethylene in D. tiedjei is catalyzed by a dehalogenase previously thought to be specific for meta-halobenzoates. PMID:16535377

  4. State of the art on nailfold capillaroscopy: a reliable diagnostic tool and putative biomarker in rheumatology?

    Science.gov (United States)

    Cutolo, Maurizio; Smith, Vanessa

    2013-11-01

    Capillaroscopy is a non-invasive and safe tool to morphologically study the microcirculation. In rheumatology it has a dual use. First, it has a role in differential diagnosis of patients with RP. Second, it may have a role in the prediction of clinical complications in CTDs. In SSc, pilot studies have shown predictive associations with peripheral vascular and lung involvement hinting at a role of capillaroscopy as putative biomarker. Also and logically, in SSc, microangiopathy, as assessed by capillaroscopy, has been associated with markers of the disease such as angiogenic/static factors and SSc-specific antibodies. Moreover, morphological assessments of the microcirculation (capillaroscopy) seem to correlate with functional assessments (such as laser Doppler). Because of its clinical and research role, eyes are geared in Europe to expand the knowledge of this tool. Both the European League Against Rheumatism (EULAR) and the ACR are stepping forward to this need.

  5. Mindfulness-Based Stress Reduction

    Science.gov (United States)

    ... R S T U V W X Y Z Mindfulness-Based Stress Reduction (MBSR) Information 6 Things You ... Disease and Dementia (12/20/13) Research Spotlights Mindfulness-Based Stress Reduction, Cognitive-Behavioral Therapy Shown To ...

  6. Alcohol harm reduction in Europe

    DEFF Research Database (Denmark)

    Herring, Rachel; Betsy, Thom; Beccaria, Franca

    2010-01-01

    The EMCDDA’s 10th scientific monograph, entitled Harm reduction: evidence, impacts and challenges provides a comprehensive overview of the harm reduction field. Part I of the monograph looks back at the emergence of harm reduction approaches and their diffusion, and explores the concept from diff...

  7. State of the direct reduction and reduction smelting processes

    Directory of Open Access Journals (Sweden)

    Markotić A.

    2002-01-01

    Full Text Available For quite a long time efforts have been made to develop processes for producing iron i.e. steel without employing conventional procedures - from ore, coke, blast furnace, iron, electric arc furnace, converter to steel. The insufficient availability and the high price of the coking coals have forced many countries to research and adopt the non-coke-consuming reduction and metal manufacturing processes (non-coke metallurgy, direct reduction, direct processes. This paper represents a survey of the most relevant processes from this domain by the end of 2000, which display a constant increase in the modern process metallurgy.

  8. Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae

    Science.gov (United States)

    Zhang, S.; Lockshin, C.; Herbert, A.; Winter, E.; Rich, A.

    1992-01-01

    A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

  9. Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp.

    Science.gov (United States)

    Deng, Peng; Tan, Xiaoqing; Wu, Ying; Bai, Qunhua; Jia, Yan; Xiao, Hong

    2015-03-01

    The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica , which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function.

  10. Cloning and sequence analysis demonstrate the chromate reduction ability of a novel chromate reductase gene from Serratia sp

    Science.gov (United States)

    DENG, PENG; TAN, XIAOQING; WU, YING; BAI, QUNHUA; JIA, YAN; XIAO, HONG

    2015-01-01

    The ChrT gene encodes a chromate reductase enzyme which catalyzes the reduction of Cr(VI). The chromate reductase is also known as flavin mononucleotide (FMN) reductase (FMN_red). The aim of the present study was to clone the full-length ChrT DNA from Serratia sp. CQMUS2 and analyze the deduced amino acid sequence and three-dimensional structure. The putative ChrT gene fragment of Serratia sp. CQMUS2 was isolated by polymerase chain reaction (PCR), according to the known FMN_red gene sequence from Serratia sp. AS13. The flanking sequences of the ChrT gene were obtained by high efficiency TAIL-PCR, while the full-length gene of ChrT was cloned in Escherichia coli for subsequent sequencing. The nucleotide sequence of ChrT was submitted onto GenBank under the accession number, KF211434. Sequence analysis of the gene and amino acids was conducted using the Basic Local Alignment Search Tool, and open reading frame (ORF) analysis was performed using ORF Finder software. The ChrT gene was found to be an ORF of 567 bp that encodes a 188-amino acid enzyme with a calculated molecular weight of 20.4 kDa. In addition, the ChrT protein was hypothesized to be an NADPH-dependent FMN_red and a member of the flavodoxin-2 superfamily. The amino acid sequence of ChrT showed high sequence similarity to the FMN reductase genes of Klebsiella pneumonia and Raoultella ornithinolytica, which belong to the flavodoxin-2 superfamily. Furthermore, ChrT was shown to have a 85.6% similarity to the three-dimensional structure of Escherichia coli ChrR, sharing four common enzyme active sites for chromate reduction. Therefore, ChrT gene cloning and protein structure determination demonstrated the ability of the gene for chromate reduction. The results of the present study provide a basis for further studies on ChrT gene expression and protein function. PMID:25667630

  11. Potential for waste reduction

    International Nuclear Information System (INIS)

    Warren, J.L.

    1990-01-01

    The author focuses on wastes considered hazardous under the Resource Conservation and Recovery Act. This chapter discusses wastes that are of interest as well as the factors affecting the quantity of waste considered available for waste reduction. Estimates are provided of the quantities of wastes generated. Estimates of the potential for waste reduction are meaningful only to the extent that one can understand the amount of waste actually being generated. Estimates of waste reduction potential are summarized from a variety of government and nongovernment sources

  12. Transmissive Olefination Route to Putative “Morinol I” Lignans

    Science.gov (United States)

    Yao, Lihua; Pitta, Bhaskar; Ravikumar, P. C.; Purzycki, Matthew; Fleming, Fraser F.

    2012-01-01

    A series of morinol-type lignans were rapidly assembled using a Grignard-based transmissive olefination. In combination with palladium-catalyzed arylations, the strategy provides stereoselective access to (7Z, 7′E), (7E, 7′E), (7E, 7′Z) morinol diastereomers and the (7Z, 8′E) and (7E, 8′E) conjugated analogs. Critical for the E/Z-stereoselectivity is a new, general method for converting alkenenitriles to alkenemethanols that circumvents the enal E/Z isomerization commonly encountered during conventional i-Bu2AlH reduction. PMID:22432777

  13. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

    Science.gov (United States)

    Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Côté, Olivier; Stare, Barbara Gerič; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

    2015-01-01

    The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

  14. Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

    Directory of Open Access Journals (Sweden)

    Shawkat Ali

    Full Text Available The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12 and an expansin-like protein (GrEXPB2, suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

  15. Reduction corporoplasty.

    Science.gov (United States)

    Hakky, Tariq S; Martinez, Daniel; Yang, Christopher; Carrion, Rafael E

    2015-01-01

    Here we present the first video demonstration of reduction corporoplasty in the management of phallic disfigurement in a 17 year old man with a history sickle cell disease and priapism. Surgical management of aneurysmal dilation of the corpora has yet to be defined in the literature. We preformed bilateral elliptical incisions over the lateral corpora as management of aneurysmal dilation of the corpora to correct phallic disfigurement. The patient tolerated the procedure well and has resolution of his corporal disfigurement. Reduction corporoplasty using bilateral lateral elliptical incisions in the management of aneurysmal dilation of the corpora is a safe an feasible operation in the management of phallic disfigurement.

  16. Microbial reductive dehalogenation.

    OpenAIRE

    Mohn, W W; Tiedje, J M

    1992-01-01

    A wide variety of compounds can be biodegraded via reductive removal of halogen substituents. This process can degrade toxic pollutants, some of which are not known to be biodegraded by any other means. Reductive dehalogenation of aromatic compounds has been found primarily in undefined, syntrophic anaerobic communities. We discuss ecological and physiological principles which appear to be important in these communities and evaluate how widely applicable these principles are. Anaerobic commun...

  17. Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

    Directory of Open Access Journals (Sweden)

    Berlin Londono-Renteria

    2015-10-01

    Full Text Available Dengue virus (DENV is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379, whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

  18. ARA-PEPs: a repository of putative sORF-encoded peptides in Arabidopsis thaliana.

    Science.gov (United States)

    Hazarika, Rashmi R; De Coninck, Barbara; Yamamoto, Lidia R; Martin, Laura R; Cammue, Bruno P A; van Noort, Vera

    2017-01-17

    Many eukaryotic RNAs have been considered non-coding as they only contain short open reading frames (sORFs). However, there is increasing evidence for the translation of these sORFs into bioactive peptides with potent signaling, antimicrobial, developmental, antioxidant roles etc. Yet only a few peptides encoded by sORFs are annotated in the model organism Arabidopsis thaliana. To aid the functional annotation of these peptides, we have developed ARA-PEPs (available at http://www.biw.kuleuven.be/CSB/ARA-PEPs ), a repository of putative peptides encoded by sORFs in the A. thaliana genome starting from in-house Tiling arrays, RNA-seq data and other publicly available datasets. ARA-PEPs currently lists 13,748 sORF-encoded peptides with transcriptional evidence. In addition to existing data, we have identified 100 novel transcriptionally active regions (TARs) that might encode 341 novel stress-induced peptides (SIPs). To aid in identification of bioactivity, we add functional annotation and sequence conservation to predicted peptides. To our knowledge, this is the largest repository of plant peptides encoded by sORFs with transcript evidence, publicly available and this resource will help scientists to effortlessly navigate the list of experimentally studied peptides, the experimental and computational evidence supporting the activity of these peptides and gain new perspectives for peptide discovery.

  19. Overall Reduction Kinetics of Low-grade Pyrolusite Using a Mixture of Hemicellulose and Lignin as Reductant

    Directory of Open Access Journals (Sweden)

    Yun-Fei Long

    2015-11-01

    Full Text Available Manganese is widely used in many fields. Many efforts have been made to recover manganese from low-grade pyrolusite due to the depletion of high-grade manganese ore. Thus, it is of practical significance to develop a clean, energy-saving and environmentally friendly technical route to reduce the low-grade pyrolusite. The reported results show that biomass wastes from crops, crop waste, wood and wood waste are environmentally friendly, energy-saving, and low-cost reducing agents for roasting reduction of low-grade pyrolusite. Kinetics of the reduction reactions is necessary for an efficient design of biomass reduction of pyrolusite. Therefore, it is important to look for a general kinetics equation to describe the reduction of pyrolusite by different kinds of biomass, because there is a wide variety of biomass wastes, meaning that it is impossible to investigate the kinetics for each biomass waste. In this paper, thermal gravimetric analysis and differential thermal analysis were applied to study the overall reduction kinetics of pyrolusite using a mixture of hemicellulose and lignin, two major components of biomass. Overall reduction process is the overlap of the respective reduction processes. A new empirical equation based on the Johnson–Mehl–Avrami equation can be used to describe the respective reduction kinetics using hemicellulose and lignin as reductants, and the corresponding apparent activation energy is 30.14 kJ mol−1 and 38.91 kJ mol−1, respectively. The overall kinetic model for the reduction of pyrolusite by the mixture of hemicellulose and lignin can be simulated by the summation of the respective kinetics by considering their mass-loss fractions, while a unit step function was used to avoid the invalid conversion data. The obtained results in this work are necessary to understand the biomass reduction of pyrolusite and provide valuable assistance in the development of a general kinetics equation.

  20. Recursions of Symmetry Orbits and Reduction without Reduction

    Directory of Open Access Journals (Sweden)

    Andrei A. Malykh

    2011-04-01

    Full Text Available We consider a four-dimensional PDE possessing partner symmetries mainly on the example of complex Monge-Ampère equation (CMA. We use simultaneously two pairs of symmetries related by a recursion relation, which are mutually complex conjugate for CMA. For both pairs of partner symmetries, using Lie equations, we introduce explicitly group parameters as additional variables, replacing symmetry characteristics and their complex conjugates by derivatives of the unknown with respect to group parameters. We study the resulting system of six equations in the eight-dimensional space, that includes CMA, four equations of the recursion between partner symmetries and one integrability condition of this system. We use point symmetries of this extended system for performing its symmetry reduction with respect to group parameters that facilitates solving the extended system. This procedure does not imply a reduction in the number of physical variables and hence we end up with orbits of non-invariant solutions of CMA, generated by one partner symmetry, not used in the reduction. These solutions are determined by six linear equations with constant coefficients in the five-dimensional space which are obtained by a three-dimensional Legendre transformation of the reduced extended system. We present algebraic and exponential examples of such solutions that govern Legendre-transformed Ricci-flat Kähler metrics with no Killing vectors. A similar procedure is briefly outlined for Husain equation.